Methodologies and compositions for creating targeted recombination and breaking linkage between traits

ABSTRACT

The current disclosure relates to methods and compositions for improving plant varieties through plant breeding and plant genetics. For instance, the disclosure concerns increasing the recombination frequency of a heterozygous trait genetically linked to a second trait within plants. Further, the disclosure concerns breaking the genetic linkage between a first allele and a second allele.

This application claims a priority based on provisional application62/352254 which was filed in the U.S. Patent and Trademark Office onJun. 20, 2016, the entire disclosure of which is hereby incorporated byreference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 31.2 KB ASCII (Text) file named “78126-US-PSP-20160620-Sequence-Listing-ST25.txt” created on Jun. 6, 2016.

BACKGROUND

Modern plant breeding relies upon traditional breeding methodologies todevelop and produce new varieties of plant species. Typically, the goalof plant breeding is to identify and selectively breed traits (as asingle gene or multiple genes) from the parental lines into progenyplants. Such processes can be limited by the genetic linkage ofdesirable traits (e.g., traits which encode a phenotype with anagronomic advantage) with undesirable traits (e.g., traits which encodea phenotype that is deleterious or exemplifies an agronomicdisadvantage).

Traditional plant breeding methodologies and compositions rely upon theanalysis of large breeding populations to identify progeny plants thathave broken linkage between a first trait located at a specific genomiclocus and a second trait located at a specific genomic locus.Especially, where the first and second traits are tightly linked in theparent plant(s). Typically, the traits, which are located at distinct,specific genomic loci, can segregate and recombine into progeny viacross-over events which rely upon breakage and rejoining of thechromosome(s). The recombination frequency between the two traits can becalculated and determined as it is known that most cross-over eventsrandomly occur along the length of the chromosome, and that therecombination frequency is a function of the distance between the firstgenomic locus that comprises a trait and second genomic locus thatcomprises a second trait. Accordingly, the closer two loci comprisingtwo traits are in physical location with one another, the less likely itis that a cross-over between the two loci comprising the two traits willoccur. The result is that the closely linked loci comprising tightlylinked traits cannot be separated from one another are transmittedtogether to the progeny. Further compounding this process is theobservation that some regions of the chromosome are characterized asgenomic locations that inherently possess low levels of recombination.Genetically linked genomic loci comprising traits that are located insuch genomic locations will remain genetically linked despite beinglocated distally from one another, thereby being transmitted together tothe progeny.

Methodologies and compositions that allow for increasing therecombination frequency between two traits located at distinct, separategenomic loci would allow for improved efficiencies in breaking thegenetic linkage between the two traits. Resultantly, development of newplant varieties could be produced more expeditiously and at less cost,in the terms of both financial and resource costs. Ultimately, the tightlinkage of desirable traits with undesirable traits can be broken sothat plant breeders can produce new plant varieties or progeny that onlycontaining the desirable traits that are desired for introgression intothe progeny by the plant breeder, and any linkage drag associated withundesirable traits is eliminated.

Therefore, a need exists for new compositions and methodologies thatallow for increasing the recombination frequency of a heterozygous traitlinked to a locus within plants, and breaking the genetic linkagebetween a first heterozygous locus comprising a trait and a second locuscomprising a trait.

BRIEF SUMMARY

In embodiments of the subject disclosure, the disclosure relates to amethod for increasing the frequency of genetic recombination between afirst locus genetically linked to a second locus within a genome of aplant, comprising: a) introducing a site specific nuclease into thegenome of the plant; b) producing a double stranded break with the sitespecific nuclease in one of two homologous chromosomes; c) undergoingrecombination within the plant genome; and, d) modifying the plantgenome, wherein the modified plant genome comprises increased frequencyof genetic recombination between the first locus and the second locus.

In an embodiment of this method, each of the loci encode at least onetrait. In further embodiments, the recombination comprises meioticrecombination or mitotic recombination. In other embodiments, thefrequency of genetic recombination is increased from 1.25 to 17.8 fold.In additional embodiments, the distance from the first locus to thesecond locus is from about 0.01 cM to about 500 cM. In furtherembodiments, the distance from the first locus to the second locus isfrom about 10 bp to about 10 Mbp. As an embodiment, the first locus islocated on a first chromosome, and the second locus is located on asecond chromosome. In an additional embodiment, the first locus to thesecond locus are present in a genomic location with low levels ofrecombination frequency. For example in an embodiment, the traitcomprises a desirable trait or an undesirable trait. In such an example,the desirable trait or the undesirable trait is either a native trait ora transgenic trait. For instance the undesirable trait can be a reducedyield, reduced resistance to disease, reduced resistance to pests,reduced tolerance to herbicide tolerance, reduced growth, reduced size,reduced production of biomass, reduced amount of produced seeds, reducedresistance against salinity, reduced resistance against heat stress,reduced resistance against cold stress, reduced resistance againstdrought stress, and any combination thereof. Alternatively, thedesirable trait can be a trait for increased yield, increased resistanceto disease, increased resistance to pests, increased tolerance toherbicides, increased growth, increased size, increased production ofbiomass, increased amount of produced seeds, increased resistanceagainst salinity, increased resistance against heat stress, increasedresistance against cold stress, increased resistance against droughtstress, and any combination thereof.

In further embodiments, the first locus comprises a polymorphic markerand the second locus comprise a trait. In an additional embodiment, thefirst locus comprises a polymorphic marker and the second locuscomprises a polymorphic marker. In further embodiments, the plant may bea polyploid plant or a diploid plant.

In subsequent embodiments, the double stranded break is produced by asite specific nuclease. For example, the site specific nuclease can be azinc finger nuclease, a TALEN nuclease, a CRISPR-Cas9 nuclease, ameganuclease, and a leucine zipper nuclease. In another embodiment, thesite specific nuclease is delivered to a cell by intra-genomicrecombination or via direct delivery.

In other embodiments, the method comprises: producing a progeny plantcomprising the modified plant genome; crossing the progeny plant withanother plant or to itself; and, generating a seed from the progenyplant. In an embodiment, the first loci comprising a first trait isheterozygous and located on the first homologous chromosome andindependently segregates from the second loci comprising a second trait,thereby resulting in a progeny plant comprising only the first locicomprising the first trait located on the first homologous chromosome.In another embodiment, the second loci comprising a second trait isheterozygous and located on the second homologous chromosome and remainsgenetically linked to a third genomic locus, thereby resulting in aprogeny plant comprising the second loci comprising the second traitgenetically linked to a third genomic locus.

In subsequent embodiments, the plant is selected from a dicotyledonousplant or a monocotyledonous plant. For example the plant may be atobacco plant, a soybean plant, a cotton plant, a Brassica plant, a cornplant, a sorghum plant, a wheat plant, or a rice plant.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: This figure shows a plasmid map of pDAB105855 containing targetDNA sequence comprising; RB7 MAR sequence/eZFN4 Binding site v1, OsUbi3promoter/Phi YFP/ZmPer5 3′UTR v2/eZFN1 binding site/ELP1 HR2 v2, ZmUbilpromoter v8/Cry34Ab1 v2/StPinII 3′ UTR v2, TaPer promoter v3/Cry35Ab1v5/StPinII 3′ UTR v2, SCBVv2/AAD-1v3/ZmLip 3′ UTR v1 between T-DNAborders.

FIG. 2: This figure shows a plasmid map of pDAB113068 containing targetDNA sequence comprising: eZFN1 Binding Site; intron from OsUbi3promoter::TraP4 DGT-28 transgene::ZmLip 3′UTR; SBS8196 BindingSite::eZFN4 Binding Site::SBS19354 Binding Site::SBS15590 BindingSite::eZFN8 Binding Site::SBS18473 Binding Site::eZFN1 Binding Site;ZmUbil promoter::PAT transgene::ZmLip 3′ between T-DNA borders.

FIG. 3: This figure shows a plasmid map of pDAB105825 containing theexpression cassette for eZFN1 site specific nuclease.

FIG. 4: This figure shows the transformation and breeding strategy usedto exemplify the increased recombination frequencies between two allelesresulting from the cleavage of one of two homologous chromosomes by asite specific nuclease (e.g., a zinc finger nuclease).

FIG. 5: This figure shows a schematic of the constructs used for thedisclosure and the location of the eZFN1 binding site.

DETAILED DESCRIPTION I. Overview

In the course of crop improvement projects involving selective breedingprotocols, it is often desirable to disrupt the genetic linkage of afirst genomic locus and a nearby second genomic locus. Especially whenthe second locus encodes a less desirable or even detrimental trait. Forinstance linkage drag may be described as the presence of geneticlinkage between two traits, for example one desirable trait and theother an undesirable trait. As a consequence of this genetic linkage thetwo loci comprising the two traits are inherited together. Reliance uponnatural recombination to generate the desirable recombinants isunsuitable if the two traits are tightly linked. Unfortunately, with themethods available in the art, removing the genetic linkage between suchdesired and undesired traits in a plant, and obtaining a plant with onlythe desired traits, has turned out to be difficult, time consuming, andin various cases impossible. If recombination frequency can be increasedduring the breeding procedure, in accordance with the methods of thepresent disclosure, recombination between the two loci comprising thetraits would be at a higher frequency in the progeny, and plant breedermay develop a new progeny plants containing only the desirable trait ina less expensive and more efficient manner.

II. Definitions

Throughout the application, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each of the twohomologous chromosomes. A diploid plant species may have differentalleles at corresponding loci on homologous chromosomes.

The term “backcross” refers to a cross between an offspring and one ofits parents or an individual genetically identical to one of itsparents. The term “backcross” encompasses “advanced backcross”, meaningcrosses between a backcross progeny and an inbred progenitor from aprior generation or an individual genetically identical to an inbredprogenitors from a prior generation. The terms “backcross” or “advancebackcross” are understood to include mating or assisted fertilization togenerate backcross progeny. Preferred methods for assisted fertilizationor reproduction include but are not limited to cloning, in vitrofertilization, or inter-cytoplasmic sperm injection. Methods forassisted fertilization are well known in the art (Thornton et al., 1999;Loutradis et al., 2000; Nakagata, 2000) U.S. Pat. Nos. 5,453,366,5,541,081, 5,849,713). Backcrossing can be used to introduce one or moresingle locus conversions from one genetic background into another.

The term “chimeric gene” (or recombinant gene) refers to any gene, whichis not normally found in nature in a species, in particular a gene inwhich one or more parts of the nucleic acid sequence are present thatare not associated with each other in nature. For example the promoteris not associated in nature with part or all of the transcribed regionor with another regulatory region. The term “chimeric gene” isunderstood to include expression constructs in which a promoter ortranscription regulatory sequence is operably linked to one or morecoding sequences or to an antisense (reverse complement of the sensestrand) or inverted repeat sequence (sense and antisense, whereby theRNA transcript forms double stranded RNA upon transcription).

The term “chromatid” refers to one of the two parts of a chromosomewhich exist after replication, there being one DNA double helix beforereplication, and two identical DNA double helices after replication, thebasic elements of the two chromatids, attached at the centromere of areplicated chromosome; intrachromosomal recombination often causes agenetic endpoint.

The term “chromosome” is one of the self-replicating, thread- orrod-shaped structures within the nuclei of eukaryotic cells thatconsists of extremely condensed chromatin and contains geneticinformation for specific functions of the cell.

The term “crossing” refers to the mating of two parent plants.

The term “crossing over” or “crossover” refers to the reciprocalexchange of chromosome arms and can, for example, be visualized at latestages of meiotic prophase I as chiasmata.

The term “desirable trait” refers to any inheritable trait that confersan advantage or increased value to a commercially cultivated crop, whilethe phrase “undesirable trait” refers to any inheritable trait which isdeleterious when expressed in the commercially cultivated crop. It isconceivable that in some cases, candidate genetic traits can becategorized as being both a “desirable trait” and an “undesirable trait”depending upon the application of the trait and the intent of the plantbreeder. Specific examples of such commercially desirable and mitigatingtraits are provided herein.

The term “diploid” refers to a typical castor plant having two sets (2N)of chromosomes, whereby each set comprises 20 chromosomes. The diploidplant, as used herein is isogenic to the multiplied polyploid planti.e., both sets of chromosomes contain essentially identical alleles inall locations. The diploid plant may be naturally occurring, geneticallymodified or a breeding product.

The term “dominant trait” refers to diploid or other polyploid organismsa trait that is phenotypically manifest in either the heterozygous ofhomozygous state, and refers to alleles that fully manifest theirphenotypic expressions over another recessive allele.

The term “expression of a gene” refers to the process wherein a DNAregion, which is operably linked to appropriate regulatory regions,particularly a promoter, is transcribed into an RNA, which isbiologically active, i.e. which is capable of being translated into abiologically active protein or peptide (or active peptide fragment) orwhich is active itself (e.g. in posttranscriptional gene silencing orRNAi). The coding sequence is preferably in sense-orientation andencodes a desired, biologically active protein or peptide, or an activepeptide fragment. In gene silencing approaches, the DNA sequence ispreferably present in the form of an antisense DNA or an inverted repeatDNA, comprising a short sequence of the target gene in antisense or insense and antisense orientation.

The term “fold change” is a measure describing how much a quantitychanges going from an initial to a final value. For example, an initialvalue of 30 and a final value of 60 corresponds to a fold change of 1(or equivalently, a change to 2 times), or in common terms, a two-foldincrease.

The term “gene” means a DNA sequence comprising a region (transcribedregion), which is transcribed into an RNA molecule (e.g. an mRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene may thus comprise several operably linked sequences, such as apromoter, a 5′ leader sequence comprising e.g. sequences involved intranslation initiation, a (protein) coding region (cDNA or genomic DNA)and a 3 non-translated sequence comprising e.g. transcriptiontermination sites.

The term “genome”, as used herein, relates to a material or mixture ofmaterials, containing genetic material from an organism. The term“genomic DNA” as used herein refers to deoxyribonucleic acids that areobtained from an organism. The terms “genome” and “genomic DNA”encompass hereditary information of an individual typically encoded innucleic acids, either DNA, or RNA, and including both genes andnon-coding sequences. The genome may refer to the nucleic acids makingup one set of chromosomes of an organism (haploid genome) or both setsof chromosomes of an organism (diploid genome) depending on the contextin which it is used.

The term “genetic recombination” used herein has a broad meaningindicating a phenomenon of DNA cleavage/rejoining involving DNAs. Themeaning of the term “genetic recombination” used in the presentdisclosure encompasses homologous recombination, non-homologousrecombination, gene conversion, inversion, unequal crossover, crossover,translocation, copy number change, chromosome fusion, and mutation. Inaddition, the term “rearrangement” refers to a situation in which theincreased frequency of “genetic recombination” causes a recombinationbetween existing genomic sequences, resulting in partial or completealteration of the genomic sequence.

The term “genetically linked” refers to a first locus being spacedwithin a given genetic distance from a second locus so that the two lociare inherited together in a progeny plant, such traits are in linkagedisequilibrium and statistically determined not to assort independently.

The term “heterozygote” or “heterozygous” means a diploid or polyploidindividual cell or plant having different alleles (forms of a givengene) at least at one locus, for instance the term denotes a geneticcondition in which different alleles reside at the same loci onhomologous chromosomes.

The term “homologous” in the context of a pair of homologous chromosomesrefers to a pair of chromosomes from an individual that are similar inlength, gene position and centromere location, and that line up andsynapse during meiosis. In an individual, one chromosome of a pair ofhomologous chromosomes comes from the mother of the individual (i.e., is“maternally-derived”), whereas the other chromosomes of the pair comesfrom the father (i.e., is “paternally-derived”). In the context ofgenes, the term “homologous” refers to a pair of genes where each generesides within each homologous chromosome at the same position and hasthe same function.

The term “homologous recombination” refers to a reciprocal exchange atcorresponding positions between between homologous chromosomes, such asbetween non-sister chromatids of homologous chromosomes during meiosis.Homologous recombination can also occur in somatic cells during mitosis(somatic crossing over).

The term “homozygote” or “homozygous” means an individual cell or planthaving the same alleles at one or more loci, for instance the termdenotes a genetic condition in which identical alleles reside at thesame loci on homologous chromosomes.

The term “intra-genomic delivery” refers to the delivery of a geneexpression cassette, wherein the gene expression cassette isincorporated within the plant genome, from one plant to a second plant(either a progeny or another parent) by crossing of the two plants.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can occur via asexual cross between two plants, where at least one of the plants hasthe desired allele within its genome. The allele is introgressed intothe progeny. In another example, transmission of an allele can occur byrecombination between two donor genomes in vitro, e.g., in a fusedprotoplast, where at least one of the donor protoplasts has the desiredallele in its genome. The desired allele can be, e.g., a transgene or aselected allele of a marker or quantitative trait locus.

The term an “isolated nucleic acid sequence” refers to a nucleic acidsequence which is no longer in the natural environment from which it wasisolated, e.g. the nucleic acid sequence in a bacterial host cell or inthe plant nuclear or plastid genome.

The term “linkage disequilibrium” refers to a non-random association ofalleles from two or more loci. It implies that a group of marker allelesor alleles have been inherited together.

The term “linkage drag” refers to the (usually undesirable) effects oftraits linked to the desirable traits being introgressed into a progenyplant. As a result of the linkage drag, the undesirable trait isinherited with the desirable trait as a result of being geneticallylinked to the undesirable trait.

The term “locus” refers to the position of a gene or any genetic sitethat has been defined genetically, for instance on a chromosome or genemap. A locus may be a gene, or part of a gene, or a DNA sequence thathas some regulatory role, and may be occupied by different sequences.The relative distance between two loci can be given by referring to theMorgan unit, but a locus can also be identified by the nature of theneighboring genes. In the methods of the subject disclosure, the firstand the second locus are different, i.e. if the first loci is at aspecific position on a first chromosome, the second loci is at aspecific position on the first chromosome which locus has a specificdistance from the first loci such that recombination frequency betweenthe loci can be determined.

The term “meiosis” or “meiotic” refers to a two-stage process of nucleardivision that reduces the somatic chromosome number (2n) to half (n) andwhich is usually followed by gamete formation. In the first stage ofmeiosis the chromosome number is reduced, wherein in the second stage ofmeiosis there is an equational division of the chromosome resulting infour daughter nuclei, each carrying one chromatid.

Although the term “mitosis” or “mitotic” is commonly used synonomouslywith the term “cell division”, mitosis correctly refers to only onephase of the cell division process: the process in which the sisterchromatids are partitioned equally between the two daughter cells. Ineukaryotic cells, mitosis is followed by cytokinesis, which is theprocess by which the cell cytoplasm is cleaved into two distinct butgenetically identical daughter cells.

The term “modify”, “modified”, “modifying” or “modification” is notespecially limited as used herein, includes an act defined by one ormore of changing, controlling, altering, attenuating, transforming, ormaking different. In one embodiment, the term “modify”, “modified”,“modifying” or “modification” includes chemical modification andbiological modification. In another embodiment, the term “modify”,“modified”, “modifying” or “modification” includes altering a plantgenome so that the genome does not contain the same original geneticmaterial as the result of deletion, substitution, additions, or otherrearrangement.

The term “morgan” or “map unit” each refer to a unit for expressing therelative distance between genes on a chromosome. One Morgan unit (cM)indicates a recombination frequency of 100%. A centimorgan (cM)indicates a recombination frequency of 1%.

The term “nucleic acid sequence” (or nucleic acid molecule) refers to aDNA or RNA molecule in single or double stranded form, particularly aDNA molecule encoding a protein or protein fragment according to thedisclosure.

The term “native trait” refers to a naturally occurring recognizednon-transgenic plant phenotype which is heritable and can be used inseveral varieties of at least one plant species. Alternatively a nativetrait is man-made and can be generated through mutagenesis of plants. Anative trait is often introgressed in a variety or plant species ofchoice by breeding. Introgression of a native trait can be carried outwith the aid of molecular markers flanking the locus or loci comprisingthe native trait of interest.

The term “phenotype” means the observable characters of an individualcell, cell culture, plant, or group of plants which results from theinteraction between that individual's genetic makeup (i.e., genotype)and the environment.

The term “plant” refers to either the whole plant or to parts of aplant, such as cells, tissue or organs (e.g. pollen, seeds, gametes,roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainablefrom the plant, as well as derivatives of any of these and progenyderived from such a plant by selfing or crossing. “Plant cell(s)”include protoplasts, gametes, suspension cultures, microspores, pollengrains, etc., either in isolation or within a tissue, organ or organism.

The term “polyploid” refers to a plant with three or more sets ofchromosomes (e.g., 3N, 4N, 5N, 6N and more). According to someembodiments of this aspect of the present disclosure, the polyploidplant is an autopolyploid.

The term “progeny” refers to the direct offspring of a particular plant(selfcross) or pair of plants (cross-pollinated) and includes all of theplants and seeds of all subsequent generations resulting from aparticular designated generation. The descendants can be, for example,of the F₁, the F₂ or any subsequent generation.

The term “promoter” refers to a nucleic acid fragment that functions tocontrol the transcription of one or more genes, located upstream withrespect to the direction of transcription of the transcriptioninitiation site of the gene, and is structurally identified by thepresence of a binding site for DNA-dependent RNA polymerase,transcription initiation sites and any other DNA sequences, including,but not limited to transcription factor binding sites, repressor andactivator protein binding sites, and any other sequences of nucleotidesknown to one of skill in the art to act directly or indirectly toregulate the amount of transcription from the promoter. A “constitutive”promoter is a promoter that is active in most tissues under mostphysiological and developmental conditions. An “inducible” promoter is apromoter that is physiologically (e.g. by external application ofcertain compounds) or developmentally regulated. A “tissue specific”promoter is only active in specific types of tissues or cells, while a“tissue preferred” promoter is preferentially, but not exclusively,active in certain tissues or cells. A “promoter which is active inplants or plant cells” is a promoter which has the capability ofinitiating transcription in plant cells.

The terms “protein” or “polypeptide” are used interchangeably and referto molecules consisting of a chain of amino acids, without reference toa specific mode of action, size, 3 dimensional structure or origin. A“fragment” or “portion” of a protein may thus still be referred to as a“protein”. An “isolated protein” is used to refer to a protein which isno longer in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell.

The term “recessive trait” refers to diploid or other polyploidorganisms a trait that is phenotypically manifest in the homozygousstate but is masked in the presence of its dominant allele.

The term “recombinant” as used herein means that something has beenrecombined, so that when made in reference to a nucleic acid constructthe term refers to a molecule that is comprised of nucleic acidsequences that are joined together or produced by means of molecularbiological techniques. The term “recombinant” or “recombination” whenmade in reference to genetic composition refers to a gamete or progenywith new combinations of alleles that did not occur in the parentalgenomes.

The term “recombination” refers to a process by which the linkage ofgenes is altered. Due to the recombination process, the combination ofgenes in a progeny molecule, cell or organism has a different patternthan the combination of genes in the parent molecules, cells ororganisms. The recombination may occur due to the exchange of DNAsequences between two chromosomes or the new association of genes in arecombinant. Within the meaning of the present disclosure, therecombination is due to the exchange of DNA sequences between twochromosomes by the process of crossing-over, i.e. the reciprocalexchange of segments of homologous chromosomes by symmetrical breakageand crosswise recombination. Hence, the terms “recombination” and“crossing-over” may be used interchangeably.

The term “recombination frequency” or “crossing-over frequency” or“frequency of genetic recombination” is used to denote the frequency bywhich crossing-over and recombination occurs between two loci on achromosome. The recombination frequency is usually calculated as thepercentage of individuals having the recombined phenotype per totalnumber of individuals analyzed. In the process of the disclosure it iscalculated as the number of individuals showing expression of thereporter protein per total number of individuals analyzed in a testcross population such as the pollen of the F1 progeny or the plants ofthe F2 progeny, as discussed further below. The “basal” recombinationfrequency is the recombination frequency in plants which are grown understandard conditions, which have not been mutagenized or treated withbiotic or abiotic stimuli and which have not been transformed withexpression cassettes other than the first and second and optionallythird and fourth expression cassette. The “induced” recombinationfrequency is the recombination frequency in plants which contain adouble strand break within one of the homologus chromosomes, therebyinfluencing recombination between the genetically linked loci.

The term “separating” means one or more process used to partially orcompletely isolate from one another one or more components, and/or oneor more process that results in one or more components being no longerlocated in the same place. The one or more components optionallyinclude, but are not limited to, one or more chromosome types, or singlechromosomes, or single copies of a chromosome type. Processes include,but are not limited to, manual, automatic, semi-automatic,remote-controlled, and/or robotic. Illustrative embodiments of suchprocesses include but are not limited to fluorescence activated cellsorting (FACS).

The term “transgenic plant” or “transformed plant” refers herein to aplant or plant cell having been transformed with a chimeric gene. Saidchimeric gene may or may not be integrated into the plant's genome. In apreferred embodiment it is not integrated. A transgenic plant cell mayrefer to a plant cell in isolation or in tissue culture, or to a plantcell contained in a plant or in a differentiated organ or tissue, andboth possibilities are specifically included herein. Hence, a referenceto a plant cell in the description or claims is not meant to refer onlyto isolated cells or protoplasts in culture, but refers to any plantcell, wherever it may be located or in whatever type of plant tissue ororgan it may be present.

The term “variety” means a subdivision of a species, consisting of agroup of individuals within the species which are distinct in form orfunction from other similar arrays of individuals.

III. Embodiments

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. In the specification, theword “comprising” is used as an open-ended term, substantiallyequivalent to the phrase “including, but not limited to”, and the word“comprises” has a corresponding meaning. Citation of references hereinshall not be construed as an admission that such references are priorart to the present invention. All publications, including but notlimited to patents and patent applications, cited in this specificationare incorporated herein by reference as if each individual publicationwere specifically and individually indicated to be incorporated byreference herein and as though fully set forth herein.

Provided are methods and compositions for increasing the frequency ofgenetic recombination between genetically linked loci within a plantgenome, wherein the loci encodes a trait, comprises an allele, orcomprises a polymorphic marker. Further provided are methods andcompositions for increasing the amount of segregation betweengenetically linked loci within a plant genome, wherein the loci encodesa trait, comprises an allele, or comprises a polymorphic marker. Alsoprovided are methods and compositions for increasing the genetic linkagedisequilibrium between genetically linked loci within a plant genome,wherein the loci encodes a trait, comprises an allele, or comprises apolymorphic marker. Generally provided are methods and compositions forreducing genetic linkage between genetically linked alleles within aplant genome, wherein the allele encodes a trait, comprises a loci, orcomprises a polymorphic marker

In an aspect, the subject disclosure relates to increasing geneticrecombination between two genomic loci. The distance between chromosomalloci is governed by recombination frequency between homologouschromosomes on which the loci are located. By monitoring the frequencyof genetic recombination between two loci, a plant breeder can determinehow many successive generations of backcrossing are required to breakthe linkage of a first locus with a second locus, so that only the firstlocus is passed onto progeny plants. In an embodiment, the geneticlinkage between two genetically linked loci is disrupted by introducinga double strand break in one of two homologous chromosomes, therebyresulting in increased genetic recombination. The increase in geneticrecombination may be determined as follows.

In an aspect, increasing the frequency of genetic recombination is anypercentage that is greater than the frequency of genetic recombinationnaturally occurring between two linked loci within a plant genome. Thefrequency of genetic recombination naturally occurring between twolinked loci within a plant genome can be determined as a percentage ofgenetic recombination. In some aspects, methods of the subjectdisclosure result to increase this frequency of genetic recombinationfrom a 1.25 fold to a 17.8 fold increase in genetic recombination. Insome embodiments of this aspect, methods to increase this frequency ofgenetic recombination result in a 1.25 fold increase in geneticrecombination. In other embodiments of this aspect, methods to increasethis frequency of genetic recombination result in a 2 fold increase ingenetic recombination. In further embodiments of this aspect, methods toincrease this frequency of genetic recombination result in a 2.25 foldincrease in genetic recombination. In embodiments of this aspect,methods to increase this frequency of genetic recombination result in a3.2 fold increase in genetic recombination. In embodiments of thisaspect, methods to increase this frequency of genetic recombinationresult in a 8 fold increase in genetic recombination. In additionalembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in a 8.6 fold increase in geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in a 13 fold increase ingenetic recombination. In embodiments of this aspect, methods toincrease this frequency of genetic recombination result in a 17.8 foldincrease in genetic recombination. In further embodiments of thisaspect, methods to increase this frequency of genetic recombinationresult in greater than a 17.75 fold increase in genetic recombination.Further increases in the frequency of genetic recombination that resultfrom methods of the subject disclosure increase genetic recombination ofgreater than a 1.25 fold, 1.5 fold, 1.75 fold, 2 fold, 2.25 fold, 2.5fold, 2.75 fold, 3 fold, 3.25 fold, 3.5 fold, 3.75 fold, 4 fold, 4.25fold, 4.5 fold, 4.75 fold, 5 fold, 5.25 fold, 5.5 fold, 5.75 fold, 6fold, 6.25 fold, 6.75 fold, 7 fold, 7.25 fold, 7.5 fold, 7.75 fold, 8fold, 8.25 fold, 8.75 fold, 9 fold, 9.25 fold, 9.5 fold, 9.75 fold, 10fold, 11 fold, 12 fold, 13 fold 14 fold, 15 fold, 16 fold, 17 fold, 18fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 30fold, 35 fold, 40 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70fold, 75 fold, 80 fold, 85 fold, 90 fold, 95 fold, or 100 fold increase.

In an aspect, increasing the frequency of genetic recombination is anypercentage that is greater than the frequency of genetic recombinationthat naturally occurs between two linked loci within a plant genome. Thefrequency of genetic recombination that naturally occurring between twolinked loci within a plant genome can be determined as a percentage ofgenetic recombination. The frequency of genetic recombination frequencycan be calculated by dividing the number of recombinant offspringcontaining only the first loci or only the second loci, alone andunlinked by the total number of offspring observed. For this disclosurethe percentage of genetic recombination that naturally occurred betweentwo linked loci within the plant genome was calculated at 3.5% as anexemplary frequency of genetic recombination. In some aspects, methodsof the subject disclosure result to increase this frequency of geneticrecombination from 4.4% to a 62.3% genetic recombination. In someembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in 4.4% genetic recombination. In otherembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in 7.1% genetic recombination. In furtherembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in 7.2% genetic recombination. Inembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in 7.9% genetic recombination. Inembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in 11.5% genetic recombination. Inadditional embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in 27.8% geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in 30.2% geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in 45.6% geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in 62.3% geneticrecombination. In some embodiments of this aspect, methods to increasethis frequency of genetic recombination result in greater than a 4.4%increase in genetic recombination. In other embodiments of this aspect,methods to increase this frequency of genetic recombination result ingreater than a 7.1% increase in genetic recombination. In furtherembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in greater than a 7.2% increase in geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in a greater than 7.9%increase in genetic recombination. In embodiments of this aspect,methods to increase this frequency of genetic recombination result ingreater than a 11.5% increase in genetic recombination. In additionalembodiments of this aspect, methods to increase this frequency ofgenetic recombination result in greater than a 27.8% increase in geneticrecombination. In embodiments of this aspect, methods to increase thisfrequency of genetic recombination result in greater than a 30.2%increase in genetic recombination. In embodiments of this aspect,methods to increase this frequency of genetic recombination result ingreater than a 45.6% genetic recombination. In embodiments of thisaspect, methods to increase this frequency of genetic recombinationresult in greater than a 62.0% increase in genetic recombination.Further increases in the frequency of genetic recombination that resultfrom methods of the subject disclosure increase genetic recombination ofgreater than 4.4%, 4.5%, 4.75%, 5%, 5.25%, 5.5%, 5.75%, 6%, 6.25%,6.75%, 7%, 7.25%, 7.5%, 7.75%, 8%, 8.25%, 8.75%, 9%, 9.25%, 9.5%, 9.75%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19, 20%, 21%, 22%, 23%,24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99%.

In another aspect, the subject disclosure relates to reducing thegenetic linkage between two linked loci. In an embodiment, the geneticlinkage between two genetically linked loci is disrupted by introducinga double strand break in one of two homologous chromosomes, therebyresulting in increased genetic recombination and a subsequent decreasein genetic linkage between two linked loci.

In another aspect, the subject disclosure relates to increasing thesegregation between two linked loci. In an embodiment, the ability oftwo genetically linked loci to segregate as only a single locus that istransmitted progeny plants is increased by introducing a double strandbreak in one of two homologous chromosomes, thereby resulting inincreased genetic recombination and a subsequent increase insegregation.

In another aspect, the subject disclosure relates to increasing thegenetic linkage disequilibrium between two linked loci. In anembodiment, the ability of two genetically linked loci to remaingenetically linked as only a single locus that is transmitted progenyplants is increased by introducing a double strand break in one of twohomologous chromosomes, thereby resulting in increased geneticrecombination and a subsequent increase in genetic linkagedisequilibrium.

In another aspect, the subject disclosure relates to recombinationoccurring cell division phase. In an embodiment recombination may occurduring meiosis. In a further embodiment recombination may occur duringmitosis. Meiosis and meiotic recombination are intricate processes whichhave been studied to different degrees and at different levels indifferent organisms. During meiosis and mitosis recombination occursbetween homologous chromosomes by crossing-over mechanisms, resulting inthe exchange of DNA segments between the homologous chromosomes.

Mitosis and meiosis are in many ways opposite processes. A principalrole of DNA recombination in mitotic cells is to preserve the fidelityof genetic information and ensure that it is faithfully reproduced andpassed on to daughter cells. In contrast, DNA recombination duringmeiosis acts to create new permutations of genetic information byfacilitating reshuffling or intermixing of the maternal and paternalgenomes during gamete formation to enable production of offspring withnovel genomes as compared to either parent. The different purposes ofDNA recombination in meiotic versus mitotic cells are reflected in thevery different rolls and mechanisms of homologous recombination in eachcell type.

There is a fundamental mechanistic distinction between the primaryprocesses of homologous recombination in meiotic (germ-line) cellscompared to mitotic (vegetative/somatic) cells. In meiotic cells,homologous recombination occurs primarily between non-sister chromatids(to shuffle the genome), whereas in mitotic cells homologousrecombination occurs primarily between sister chromatids (to correctgenomic errors). Sister chromatids are replicated copies of a particularmaternal or paternal chromosome. Recombination between non-sisterchromatids (i.e. between a paternal chromatid and a maternal chromatid)occurs 500-1000 fold more frequently in meiotic cells versus mitoticcells. The meiotic process of non-sister chromatid exchange (NSCE)facilitates novel recombination of the genetic information from twoparents of the organism. In contrast, the mitotic process ofsister-chromatid exchange (SCE) resulting from recombination-mediatedrepair is a primary mechanism for maintaining genome fidelity throughouta multi-cellular organism.

In specific embodiments, the recombination occurs during a cytologicalphase of meiosis. During meiosis a number of cytological phases aredistinguished and for each phase a number of mutants has been describedin plants. During the initial phase called meiotic Prophase a number ofstages are discerned. During the initial stage called Leptotene, theindividual chromosomes which have been replicated and which consist oftwo sister chromatids start to condense and become shorter and thicker.Simultaneously, the nuclear envelope starts to disintegrate and thehomologous chromosomes start to associate. The next stage is calledZygotene in which the chromosomes are fully condensed and in which thehomologous chromosomes align and start to form the so-calledsynaptonemal complex (SC). The difl/synl mutant of Arabidopsis isimpaired in the formation of the SC (Bhatt, A. M. et al (1999) Plant J.19, 463-472; Bal, X. et al (1999) Plant Cell 11, 417-430). The DIF1/SYN1gene products are homologous to the yeast cohesin REC8/RAD21 whichfunction in synapsis and recombination. At Pachytene the formation ofthe SC is completed for all chromosomes. At this stage meioticrecombination occurs which is initiated by the formation ofdouble-stranded breaks followed by chromatid exchange between homologouschromosomes. The physical links that are established between thenon-sister chromatids and which persist even in the absence of thesynaptonemal complex are called chiasmata. During Diplotene andDiakinesis the chromosomes fully condense, the nuclear envelope hasdisappeared and the spindle fibers have been formed. Subsequently duringMetaphase I, the pairs of homologous chromosomes are located in theequatorial plane of the cell. Then, during Anaphase I, the homologouschromosomes, each consisting of two sister chromatids which may haveundergone a number of recombination events and are held together by acentromere, move towards the opposite cellular poles. During TelophaseI, the polar movement is completed, the spindle disappears and the cellstarts to divide.

Subsequently, these cells enter Prophase II that is characterized by thealignment of the condensed chromosomes on the equatorial plane. Aspindle complex is being formed. During Metaphase II the chromosomes arefully aligned at the equatorial plane and the spindle complex iscompleted. During the next phase, called Anaphase II, the centromeresdivide and the sister chromatids move towards opposite poles. InTelophase II this movement process is completed, the spindle complexstarts disappearing and cell division initiates. Subsequently, thechromosomes resume their Interphase appearance characterized by uncoiledchromosomes located inside the nuclear envelope.

The end product of meiosis II is a set of four genetically distincthaploid cells, which can undergo mitosis to develop into gametophytes.The gametophytes produce the gametes, which upon fusion leads to theformation of a zygote, which develops, into an embryo that can grow outinto the next generation sporophyte.

The genetic variation, which occurs in the sporophyte, is determined bythe genotypes of the female and male gametes that fused upon theformation of the zygote. Therefore this genetic variation is createdduring the formation of the female and male spores during meiosis whichleads to genetic re-assortment of the original parental chromosomes aswell as chromosomal regions due to recombination events.

In an aspect, the disclosure relates to genetically linked loci.Generally, a locus is any polynucleotide sequence that is physicallylocated on a chromosome. As an embodiment, the locus can span any rangeof polynucleotide base pairs from 1 base pair to 1,0000,000 base pairs(i.e., 1 Mbp) in length. In a further embodiment, the locus may comprisean allele. In another embodiment, the locus may comprise a trait. In anadditional embodiment, the locus may comprise a polymorphic marker.

In further embodiments, a trait can include a transgenic trait.Transgenic traits that are suitable for use in the present disclosedconstructs include, but are not limited to, coding sequences that confer(1) resistance to pests or disease, (2) tolerance to herbicides, (3)value added agronomic traits, such as; yield improvement, nitrogen useefficiency, water use efficiency, and nutritional quality, (4) bindingof a protein to DNA in a site specific manner, (5) expression of smallRNA, and (6) selectable markers. In accordance with one embodiment, thetransgene encodes a selectable marker or a gene product conferringinsecticidal resistance, herbicide tolerance, small RNA expression,nitrogen use efficiency, water use efficiency, or nutritionalquality. 1. Insect Resistance

Various insect resistance coding sequences are an embodiment of atransgenic trait. The operably linked sequences can then be incorporatedinto a chosen vector to allow for identification and selection oftransformed plants (“transformants”). Exemplary insect resistance codingsequences are known in the art. As embodiments of insect resistancecoding sequences that can be operably linked to the regulatory elementsof the subject disclosure, the following traits are provided. Codingsequences that provide exemplary Lepidopteran insect resistance include:cry1A; cry1A.105; cry1Ab; cry1Ab(truncated); cry1Ab-Ac (fusion protein);cry1Ac (marketed as Widestrike®); cry1C; cry1F (marketed asWidestrike®); cry1Fa2; cry2Ab2; cry2Ae; cry9C; mocrylF;pinII (proteaseinhibitor protein); vip3A(a); and vip3Aa20. Coding sequences thatprovide exemplary Coleopteran insect resistance include: cry34Ab1(marketed as Herculex®); cry35Ab1 (marketed as Herculex®); cry3A;cry3Bb1; dvsnf7; and mcry3A. Coding sequences that provide exemplarymulti-insect resistance include ecry31.Ab. The above list of insectresistance genes is not meant to be limiting. Any insect resistancegenes are encompassed by the present disclosure.

2. Herbicide Tolerance

Various herbicide tolerance coding sequences are an embodiment of atransgenic trait. Exemplary herbicide tolerance coding sequences areknown in the art. As embodiments of herbicide tolerance coding sequencesthat can be operably linked to the regulatory elements of the subjectdisclosure, the following traits are provided. The glyphosate herbicidecontains a mode of action by inhibiting the EPSPS enzyme(5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involvedin the biosynthesis of aromatic amino acids that are essential forgrowth and development of plants. Various enzymatic mechanisms are knownin the art that can be utilized to inhibit this enzyme. The genes thatencode such enzymes can be operably linked to the gene regulatoryelements of the subject disclosure. In an embodiment, selectable markergenes include, but are not limited to genes encoding glyphosateresistance genes include: mutant EPSPS genes such as 2mEPSPS genes, cp4EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosatedegradation genes such as glyphosate acetyl transferase genes (gat) andglyphosate oxidase genes (gox). These traits are currently marketed asGly-Tol™, Optimum® GAT®, Agrisure® GT and Roundup Ready®. Resistancegenes for glufosinate and/or bialaphos compounds include dsm-2, bar andpat genes. The bar and pat traits are currently marketed asLibertyLink®. Also included are tolerance genes that provide resistanceto 2,4-D such as aad-1 genes (it should be noted that aad-1 genes havefurther activity on arloxyphenoxypropionate herbicides) and aad-12 genes(it should be noted that aad-12 genes have further activity onpyidyloxyacetate synthetic auxins). These traits are marketed as Enlist®crop protection technology. Resistance genes for ALS inhibitors(sulfonylureas, imidazolinones, triazolopyrimidines,pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) areknown in the art. These resistance genes most commonly result from pointmutations to the ALS encoding gene sequence. Other ALS inhibitorresistance genes include hra genes, the csr1-2 genes, Sr-HrA genes, andsurB genes. Some of the traits are marketed under the tradenameClearfield®. Herbicides that inhibit HPPD include the pyrazolones suchas pyrazoxyfen, benzofenap, and topramezone; triketones such asmesotrione, sulcotrione, tembotrione, benzobicyclon; and diketonitrilessuch as isoxaflutole. These exemplary HPPD herbicides can be toleratedby known traits. Examples of HPPD inhibitors include hppdPF_W336 genes(for resistance to isoxaflutole) and avhppd-03 genes (for resistance tomeostrione). An example of oxynil herbicide tolerant traits include thebxn gene, which has been showed to impart resistance to theherbicide/antibiotic bromoxynil. Resistance genes for dicamba includethe dicamba monooxygenase gene (dmo) as disclosed in International PCTPublication No. WO 2008/105890. Resistance genes for PPO or PROTOXinhibitor type herbicides (e.g., acifluorfen, butafenacil, flupropazil,pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen,azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen,fomesafen, fluoroglycofen, and sulfentrazone) are known in the art.Exemplary genes conferring resistance to PPO include over expression ofa wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B,(2000) Overexpression of plastidic protoporphyrinogen IX oxidase leadsto resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol122:75-83.), the B. subtilis PPO gene (Li, X. and Nicholl D. 2005.Development of PPO inhibitor-resistant cultures and crops. Pest Manag.Sci. 61:277-285 and Choi K W, Han O, Lee H J, Yun Y C, Moon Y H, Kim MK, Kuk Y I, Han S U and Guh J O, (1998) Generation of resistance to thediphenyl ether herbicide, oxyfluorfen, via expression of the Bacillussubtilis protoporphyrinogen oxidase gene in transgenic tobacco plants.Biosci Biotechnol Biochem 62:558-560.) Resistance genes for pyridinoxyor phenoxy proprionic acids and cyclohexones include the ACCaseinhibitor-encoding genes (e.g., Acc1-S1, Accl-S2 and Accl-S3). Exemplarygenes conferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid include haloxyfop, diclofop, fenoxyprop,fluazifop, and quizalofop. Finally, herbicides can inhibitphotosynthesis, including triazine or benzonitrile are providedtolerance by psbA genes (tolerance to triazine), 1s+ genes (tolerance totriazine), and nitrilase genes (tolerance to benzonitrile). The abovelist of herbicide tolerance genes is not meant to be limiting. Anyherbicide tolerance genes are encompassed by the present disclosure.

3. Agronomic Traits

Various agronomic trait coding sequences are an embodiment of atransgenic trait. Exemplary agronomic trait coding sequences are knownin the art. As embodiments of agronomic trait coding sequences that canbe operably linked to the regulatory elements of the subject disclosure,the following traits are provided. Delayed fruit softening as providedby the pg genes inhibit the production of polygalacturonase enzymeresponsible for the breakdown of pectin molecules in the cell wall, andthus causes delayed softening of the fruit. Further, delayed fruitripening/senescence of acc genes act to suppress the normal expressionof the native acc synthase gene, resulting in reduced ethyleneproduction and delayed fruit ripening. Whereas, the accd genesmetabolize the precursor of the fruit ripening hormone ethylene,resulting in delayed fruit ripening. Alternatively, the sam-k genescause delayed ripening by reducing S-adenosylmethionine (SAM), asubstrate for ethylene production. Drought stress tolerance phenotypesas provided by cspB genes maintain normal cellular functions under waterstress conditions by preserving RNA stability and translation. Anotherexample includes the EcBetA genes that catalyze the production of theosmoprotectant compound glycine betaine conferring tolerance to waterstress. In addition, the RmBetA genes catalyze the production of theosmoprotectant compound glycine betaine conferring tolerance to waterstress. Photosynthesis and yield enhancement is provided with the bbx32gene that expresses a protein that interacts with one or more endogenoustranscription factors to regulate the plant's day/night physiologicalprocesses. Ethanol production can be increase by expression of theamy797E genes that encode a thermostable alpha-amylase enzyme thatenhances bioethanol production by increasing the thermostability ofamylase used in degrading starch. Finally, modified amino acidcompositions can result by the expression of the cordapA genes thatencode a dihydrodipicolinate synthase enzyme that increases theproduction of amino acid lysine. The above list of agronomic traitcoding sequences is not meant to be limiting. Any agronomic trait codingsequence is encompassed by the present disclosure.

4. DNA Binding Proteins

Various DNA binding protein coding sequences are an embodiment of atransgenic trait. Exemplary DNA binding protein coding sequences areknown in the art. As embodiments of DNA binding protein coding sequencesthat can be operably linked to the regulatory elements of the subjectdisclosure, the following types of DNA binding proteins can include;Zinc Fingers, Talens, CRISPRS, and meganucleases. The above list of DNAbinding protein coding sequences is not meant to be limiting. Any DNAbinding protein coding sequences is encompassed by the presentdisclosure.

5. Small RNA

Various small RNAs are an embodiment of a transgenic trait. Exemplarysmall RNA traits are known in the art. As embodiments of small RNAcoding sequences that can be operably linked to the regulatory elementsof the subject disclosure, the following traits are provided. Forexample, delayed fruit ripening/senescence of the anti-efe small RNAdelays ripening by suppressing the production of ethylene via silencingof the ACO gene that encodes an ethylene-forming enzyme. The alteredlignin production of ccomt small RNA reduces content of guanacyl (G)lignin by inhibition of the endogenous S-adenosyl-L-methionine:trans-caffeoyl CoA 3-O-methyltransferase (CCOMT gene). Further, theBlack Spot Bruise Tolerance in Solanum verrucosum can be reduced by thePpo5 small RNA which triggers the degradation of Ppo5 transcripts toblock black spot bruise development. Also included is the dvsnf7 smallRNA that inhibits Western Corn Rootworm with dsRNA containing a 240 bpfragment of the Western Corn Rootworm Snf7 gene. Modifiedstarch/carbohydrates can result from small RNA such as the pPhL smallRNA (degrades PhL transcripts to limit the formation of reducing sugarsthrough starch degradation) and pR1 small RNA (degrades R1 transcriptsto limit the formation of reducing sugars through starch degradation).Additional, benefits such as reduced acrylamide resulting from the asnlsmall RNA that triggers degradation of Asn1 to impair asparagineformation and reduce polyacrylamide. Finally, the non-browning phenotypeof pgas ppo suppression small RNA results in suppressing PPO to produceapples with a non-browning phenotype. The above list of small RNAs isnot meant to be limiting. Any small RNA encoding sequences areencompassed by the present disclosure.

6. Selectable Markers

Various selectable markers also described as reporter genes are anembodiment of a transgenic trait. Many methods are available to confirmexpression of selectable markers in transformed plants, including forexample DNA sequencing and PCR (polymerase chain reaction), Southernblotting, RNA blotting, immunological methods for detection of a proteinexpressed from the vector. But, usually the reporter genes are observedthrough visual observation of proteins that when expressed produce acolored product. Exemplary reporter genes are known in the art andencode β-glucuronidase (GUS), luciferase, green fluorescent protein(GFP), yellow fluorescent protein (YFP, Phi-YFP), red fluorescentprotein (DsRFP, RFP, etc), β-galactosidase, and the like (See Sambrook,et al., Molecular Cloning: A Laboratory Manual, Third Edition, ColdSpring Harbor Press, N.Y., 2001, the content of which is incorporatedherein by reference in its entirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO),spectinomycin/streptinomycin resistance (AAD), and hygromycinphosphotransferase (HPT or HGR) as well as genes conferring resistanceto herbicidal compounds. Herbicide resistance genes generally code for amodified target protein insensitive to the herbicide or for an enzymethat degrades or detoxifies the herbicide in the plant before it canact. For example, resistance to glyphosate has been obtained by usinggenes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutantsfor EPSPS are well known, and further described below. Resistance toglufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D)have been obtained by using bacterial genes encoding PAT or DSM-2, anitrilase, an AAD-1, or an AAD-12, each of which are examples ofproteins that detoxify their respective herbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar and pat genes from Streptomycesspecies, including Streptomyces hygroscopicus and Streptomycesviridichromogenes, and pyridinoxy or phenoxy proprionic acids andcyclohexones (ACCase inhibitor-encoding genes). Exemplary genesconferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid (including haloxyfop, diclofop, fenoxyprop,fluazifop, quizalofop) include genes of acetyl coenzyme A carboxylase(ACCase); Acc1-S1, Accl-S2 and Accl-S3. In an embodiment, herbicides caninhibit photosynthesis, including triazine (psbA and 1s+ genes) orbenzonitrile (nitrilase gene). Futhermore, such selectable markers caninclude positive selection markers such as phosphomannose isomerase(PMI) enzyme.

In an embodiment, selectable marker genes include, but are not limitedto genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamidehydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA). An embodiment also includesselectable marker genes encoding resistance to: chloramphenicol;methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; andphosphinothricin. The above list of selectable marker genes is not meantto be limiting. Any reporter or selectable marker gene are encompassedby the present disclosure.

In some embodiments the coding sequences are synthesized for optimalexpression in a plant. For example, in an embodiment, a coding sequenceof a gene has been modified by codon optimization to enhance expressionin plants. An insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, ora selectable marker transgene can be optimized for expression in aparticular plant species or alternatively can be modified for optimalexpression in dicotyledonous or monocotyledonous plants. Plant preferredcodons may be determined from the codons of highest frequency in theproteins expressed in the largest amount in the particular plant speciesof interest. In an embodiment, a coding sequence, gene, or transgene isdesigned to be expressed in plants at a higher level resulting in highertransformation efficiency. Methods for plant optimization of genes arewell known. Guidance regarding the optimization and production ofsynthetic DNA sequences can be found in, for example, WO2013016546,WO2011146524, WO1997013402, U.S. Pat. Nos. 6,166,302, and 5,380,831,herein incorporated by reference.

In further embodiments, a trait can include a non-transgenic trait, suchas a native trait or an endogenous trait. Exemplary native traits caninclude yield traits, resistance to disease traits, resistance to peststraits, tolerance to herbicide tolerance traits, growth traits, sizetraits, production of biomass traits, amount of produced seeds traits,resistance against salinity traits, resistance against heat stresstraits, resistance against cold stress traits, resistance againstdrought stress traits, male sterility traits, waxy starch traits,modified fatty acid metabolism traits, modified phytic acid metabolismtraits, modified carbohydrate metabolism traits, modified proteinmetabolism traits, and any combination of such traits.

In further embodiments, exemplary native traits can include early vigor,stress tolerance, drought tolerance, increased nutrient use efficiency,increased root mass and increased water use efficiency. Additionalexemplary native traits can include resistance to fungal, bacterial andviral pathogens, plant insect resistance; modified flower size, modifiedflower number, modified flower pigmentation and shape, modified leafnumber, modified leaf pigmentation and shape, modified seed number,modified pattern or distribution of leaves and flowers, modified stemlength between nodes, modified root mass and root developmentcharacteristics, and increased drought, salt and antibiotic tolerance.Fruit-specific native traits include modified lycopene content, modifiedcontent of metabolites derived from lycopene including carotenes,anthocyanins and xanthophylls, modified vitamin A content, modifiedvitamin C content, modified vitamin E content, modified fruitpigmentation and shape, modified fruit ripening characteristics, fruitresistance to fungal, bacterial and viral pathogens, fruit resistance toinsects, modified fruit size, and modified fruit texture, e.g., solublesolids, total solids, and cell wall components.

In an aspect, the native traits may be specific to a particular crop.Exemplary native traits in corn can include the traits described in U.S.Pat. No. 9,288,955, herein incorporated by reference in its entirety.Exemplary native traits in soybean can include the traits described inU.S. Pat. No. 9,313,978, herein incorporated by reference in itsentirety. Exemplary native traits in cotton can include the traitsdescribed in U.S. Pat. No. 8,614,375, herein incorporated by referencein its entirety. Exemplary native traits in sorghum can include thetraits described in U.S. Pat. No. 9,080,182, herein incorporated byreference in its entirety. Exemplary native traits in wheat can includethe traits described in U.S. Patent Application No. 2015/0040262, hereinincorporated by reference in its entirety. Exemplary native traits inwheat can include the traits described in U.S. Pat. No. 8,927,833,herein incorporated by reference in its entirety. Exemplary nativetraits in Brassica plants can include the traits described in U.S. Pat.No. 8,563,810, herein incorporated by reference in its entirety.Exemplary native traits in tobacco plants can include the traitsdescribed in U.S. Pat. No. 9,096,864, herein incorporated by referencein its entirety.

In a further aspect, exemplary polymorphic markers can include geneticmarker profiles obtained by techniques such as restriction fragmentlength polymorphisms (RFLPs), randomly amplified polymorphic DNAs(RAPDs), arbitrarily primed polymerase chain reaction (AP-PCR), DNAamplification fingerprinting (DAF), sequence characterized amplifiedregions (SCARs), amplified fragment length polymorphisms (AFLPs), simplesequence repeats (SSRs) also referred to as microsatellites, or singlenucleotide polymorphisms (SNPs). For example, see Cregan et al. (1999)“An Integrated Genetic Linkage Map of the Soybean Genome” Crop Science39:1464-1490, and Berry et al. (2003) “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize InbredLines and Soybean Varieties” Genetics 165:331-342, each of which areincorporated by reference herein in their entirety.

Genetic maps defined with polymorphic markers may also be used with thedisclosed method. This process of marker assisted selection or markerassisted breeding involves linking polymorphic markers to a desiredtrait or allele by determining the frequency of co-inheritance ofpolymorphic markers with the trait/allele. Once a polymorphic markershas been found which is inherited at high frequency with the targettrait, it can be used as a molecular probe to screen a DNA library ofthe organism to identify a fragment of DNA which encodes the cognategene or quantitative trait loci. One major difficulty with markerassisted selection or marker assisted breeding is that the relationshipbetween genetic distance and physical distance can vary between speciesand even between different regions of the genome in a given species.Therefore a molecular marker may show absolute linkage to the targettrait locus but it may be physically hundreds of kilobases away from theactual gene of interest. This makes identifying and introgressing theactual gene responsible for the trait difficult because there may bevast stretches of DNA to evaluate in order to identify the trait/allele.In addition, one might map more than one polymorphic marker showingabsolute linkage to the target trait locus. However, using a reasonablepopulation size, it may not be possible to identify which marker isphysically closer to the target gene. Thus, while one marker may be 10kilobases from the target gene and the other is 400 kilobases from thetarget gene, with conventional methods relying on natural levels ofrecombination frequency there may be no way of differentiating which ofthe two markers should be used to most efficiently clone the targetgene. It would therefore be beneficial to map-based cloning projects toutilize the present method of the subject disclosure to provide elevatedrecombination levels so as to increase precision in determining geneticdistance between polymorphic markers and target trait loci.

Means of performing genetic polymorphic marker profiles usingpolymorphisms are well known in the art. SSRs, RFLPs, RAPDs, AP-PCT,DAF, SCARs, AFLPs, and SNPs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. Thepolymorphisms may refers to single nucleotide exchanges, or di-, tri- ortetra-nucleotide repeats within a genome. The repeat region may vary inlength between genotypes while the DNA flanking the repeat is conservedsuch that the primers will work in a plurality of genotypes. Apolymorphism between two genotypes represents repeats of differentlengths between the two flanking conserved DNA sequences. A markersystem based on SSRs, RFLPs, RAPDs, AP-PCT, DAF, SCARs, AFLPs, or SNPscan be highly informative in linkage analysis relative to other markersystems in that multiple alleles may be present. Another advantage ofthis type of marker is that, through use of flanking primers, detectionof polymorphic markers can be achieved, for example, by the polymerasechain reaction (PCR). The PCR detection is done by the use of twooligonucleotide primers flanking the polymorphic segment of thepolymorphism followed by DNA amplification. This step involves repeatedcycles of heat denaturation of the DNA followed by annealing of theprimers to their complementary sequences at low temperatures, andextension of the annealed primers with DNA polymerase. Size separationof DNA fragments on agarose or polyacrylamide gels followingamplification, comprises the major part of the methodology. Suchselection and screening methodologies are well known to those skilled inthe art. Molecular confirmation methods that can be used to identifytransgenic plants are known to those with skill in the art. Severalexemplary methods are further described below.

Molecular Beacons have been described for use in sequence detection,such as a polymorphic sequence, a trait, or an allele. Briefly, a FREToligonucleotide probe is designed that overlaps the flanking genomic andinsert DNA junction. The unique structure of the FRET probe results init containing a secondary structure that keeps the fluorescent andquenching moieties in close proximity. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Following successful PCR amplification, hybridization of the FRETprobe(s) to the target sequence results in the removal of the probesecondary structure and spatial separation of the fluorescent andquenching moieties. A fluorescent signal indicates the presence of theflanking genomic/transgene insert sequence due to successfulamplification and hybridization. Such a molecular beacon assay fordetection of as an amplification reaction is an embodiment of thesubject disclosure.

Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies,Foster City, Calif.), is a method of detecting and quantifying thepresence of a DNA sequence, such as a polymorphic sequence, a trait, oran allele. Briefly, a FRET oligonucleotide probe is designed with oneoligo within the transgene and one in the flanking genomic sequence forevent-specific detection. The FRET probe and PCR primers (one primer inthe insert DNA sequence and one in the flanking genomic sequence) arecycled in the presence of a thermostable polymerase and dNTPs.Hybridization of the FRET probe results in cleavage and release of thefluorescent moiety away from the quenching moiety on the FRET probe. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization. Sucha hydrolysis probe assay for detection of as an amplification reactionis an embodiment of the subject disclosure.

KASPar® assays are a method of detecting and quantifying the presence ofa DNA sequence, such as a polymorphic sequence, a trait, or an allele.Briefly, the genomic DNA sample comprising the integrated geneexpression cassette polynucleotide is screened using a polymerase chainreaction (PCR) based assay known as a KASPar® assay system. The KASPar®assay used in the practice of the subject disclosure can utilize aKASPar® PCR assay mixture which contains multiple primers. The primersused in the PCR assay mixture can comprise at least one forward primersand at least one reverse primer. The forward primer contains a sequencecorresponding to a specific region of the DNA polynucleotide, and thereverse primer contains a sequence corresponding to a specific region ofthe genomic sequence. In addition, the primers used in the PCR assaymixture can comprise at least one forward primers and at least onereverse primer. For example, the KASPar® PCR assay mixture can use twoforward primers corresponding to two different alleles and one reverseprimer. One of the forward primers contains a sequence corresponding tospecific region of the endogenous genomic sequence. The second forwardprimer contains a sequence corresponding to a specific region of the DNApolynucleotide. The reverse primer contains a sequence corresponding toa specific region of the genomic sequence. Such a KASPar® assay fordetection of an amplification reaction is an embodiment of the subjectdisclosure.

In some embodiments the fluorescent signal or fluorescent dye isselected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

In other embodiments the amplification reaction is run using suitablesecond fluorescent DNA dyes that are capable of staining cellular DNA ata concentration range detectable by flow cytometry, and have afluorescent emission spectrum which is detectable by a real timethermocycler. It should be appreciated by those of ordinary skill in theart that other nucleic acid dyes are known and are continually beingidentified. Any suitable nucleic acid dye with appropriate excitationand emission spectra can be employed, such as YO-PRO-1®, SYTOX Green®,SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®.

In further embodiments, Next Generation Sequencing (NGS) can be used fordetection of a sequence, such as a polymorphic sequence, a trait, or anallele. As described by Brautigma et al., 2010, DNA sequence analysiscan be used to determine the nucleotide sequence of the isolated andamplified fragment. The amplified fragments can be isolated andsub-cloned into a vector and sequenced using chain-terminator method(also referred to as Sanger sequencing) or Dye-terminator sequencing. Inaddition, the amplicon can be sequenced with Next Generation Sequencing.NGS technologies do not require the sub-cloning step, and multiplesequencing reads can be completed in a single reaction. Three NGSplatforms are commercially available, the Genome Sequencer FLX™ from 454Life Sciences/Roche, the Illumina Genome Analyser™ from Solexa andApplied Biosystems' SOLiD™ (acronym for: ‘Sequencing by Oligo Ligationand Detection’). In addition, there are two single molecule sequencingmethods that are currently being developed. These include the trueSingle Molecule Sequencing (tSMS) from Helicos Bioscience™ and theSingle Molecule Real Time™ sequencing (SMRT) from Pacific Biosciences.

The Genome Sequencher FLX™ which is marketed by 454 Life Sciences/Rocheis a long read NGS, which uses emulsion PCR and pyrosequencing togenerate sequencing reads. DNA fragments of 300-800 bp or librariescontaining fragments of 3-20 kb can be used. The reactions can produceover a million reads of about 250 to 400 bases per run for a total yieldof 250 to 400 megabases. This technology produces the longest reads butthe total sequence output per run is low compared to other NGStechnologies.

The Illumina Genome Analyser™ which is marketed by Solexa™ is a shortread NGS which uses sequencing by synthesis approach with fluorescentdye-labeled reversible terminator nucleotides and is based onsolid-phase bridge PCR. Construction of paired end sequencing librariescontaining DNA fragments of up to 10 kb can be used. The reactionsproduce over 100 million short reads that are 35-76 bases in length.This data can produce from 3-6 gigabases per run.

The Sequencing by Oligo Ligation and Detection (SOLiD) system marketedby Applied Biosystems™ is a short read technology. This NGS technologyuses fragmented double stranded DNA that are up to 10 kb in length. Thesystem uses sequencing by ligation of dye-labelled oligonucleotideprimers and emulsion PCR to generate one billion short reads that resultin a total sequence output of up to 30 gigabases per run.

tSMS of Helicos Bioscience™ and SMRT of Pacific Biosciences™ apply adifferent approach which uses single DNA molecules for the sequencereactions. The tSMS Helicos™ system produces up to 800 million shortreads that result in 21 gigabases per run. These reactions are completedusing fluorescent dye-labelled virtual terminator nucleotides that isdescribed as a ‘sequencing by synthesis’ approach.

The SMRT Next Generation Sequencing system marketed by PacificBiosciences™ uses a real time sequencing by synthesis. This technologycan produce reads of up to 1,000 bp in length as a result of not beinglimited by reversible terminators. Raw read throughput that isequivalent to one-fold coverage of a diploid human genome can beproduced per day using this technology.

In further embodiments, exemplary alleles are included in the subjectdisclosure. A polymorphism is thus said to be allelic, in that, due tothe existence of the polymorphism, some members of a species may havethe “standard” sequence (i.e. the standard “allele”) whereas othermembers may have a variant sequence (i.e., a variant “allele”). Thus, asused herein, an allele is one of two or more alternative versions of agene or other genetic region at a particular location on a chromosome.In the simplest case, only one variant sequence may exist, and thepolymorphism is thus said to be bi-allelic. In other cases, the species'population may contain multiple alleles, and the polymorphism is termedtri-allelic, etc.

A single gene encoding trait or genetic region may have multipledifferent unrelated polymorphisms. For example, it may have a onebi-allelic polymorphism at one site, another bi-allelic polymorphism atanother site and a multi-allelic polymorphism at another site. When allthe sequences for a group of alleles at a chromosomal locus in a plantare the same, the alleles are said to be homozygous at that locus. Whenthe sequence of any allele at a particular locus in a plant isdifferent, the population of alleles is said to be heterozygous at thatlocus. In an embodiment, the locus may be present in the plant genome asa heterozygous locus. In a further embodiment, the locus may becomprised of a trait that is present in the plant genome as aheterozygous trait. In a further embodiment, the locus may be comprisedof an allele that is present in the plant genome as a heterozygousallele. In a further embodiment, the locus may be comprised of apolymorphic marker that is present in the plant genome as a heterozygouspolymorphic marker.

In another aspect, the subject disclosure relates to desirable traits.In an embodiment, the desirable trait may be closely linked to anotherundesirable trait. The plant breeder decides which trait is desirableand which trait is undesirable. Generally, it is the goal of the plantbreeder to produce a progeny plant that possess the desirable trait(s)and does not possess the undesirable trait(s). The methods of thedisclosure provide a solution to overcome the tight genetic linkagebetween two such traits (i.e., a desirable trait genetically linked toan undesirable trait). The genetic linkage of a desirable trait to anundesirable trait results in the inheritance of the two traits intoprogeny plants, and is commonly referred to as linkage drag. In anembodiment, the desirable trait may be a native trait. In a furtherembodiment, the desirable trait may be a transgenic trait. In anembodiment, the undesirable trait may be a native trait. In anotherembodiment, the undesirable trait may be a transgenic trait.

Exemplary undesirable traits can include reduced yield traits, reducedresistance to disease traits, reduced resistance to pests traits,reduced tolerance to herbicide tolerance traits, reduced growth traits,reduced size traits, reduced production of biomass traits, reducedamount of produced seeds traits, reduced resistance against salinitytraits, reduced resistance against heat stress traits, reducedresistance against cold stress traits, reduced resistance againstdrought stress traits, male sterility traits, waxy starch traits,modified fatty acid metabolism traits, modified phytic acid metabolismtraits, modified carbohydrate metabolism traits, modified proteinmetabolism traits, and any combination of such traits.

Exemplary desirable traits can include increased yield traits, increasedresistance to disease traits, increased resistance to pests traits,increased tolerance to herbicide tolerance traits, increased growthtraits, increased size traits, increased production of biomass traits,increased amount of produced seeds traits, increased resistance againstsalinity traits, increased resistance against heat stress traits,increased resistance against cold stress traits, increased resistanceagainst drought stress traits, male sterility traits, waxy starchtraits, modified fatty acid metabolism traits, modified phytic acidmetabolism traits, modified carbohydrate metabolism traits, modifiedprotein metabolism traits, and any combination of such traits.

In another aspect, the subject disclosure relates to a method to selectrecessive traits and to segregate these traits away from dominant traitsin progeny plants. In an embodiment, the recessive trait may be presentin a plant genome as a heterozygous trait with a dominant trait. Theplant breeder may decide to produce progeny plants that possess therecessive trait. The methods of the disclosure provide a solution topass the recessive trait into progeny plants. For example, a doublestrand break may be specifically introduced into the dominant trait sothat only the recessive trait is passed to the progeny plants. In anembodiment, a first allele may encode a dominant trait and a secondallele may encode a recessive trait. In further embodiments, a method tointroduce a double strand break in the dominant trait results in theproduction of progeny plants that contain the recessive trait.

In a further aspect, the subject disclosure relates to a method toselect dominant traits and to segregate these traits away from recessivetraits in progeny plants. In an embodiment, the dominant trait may bepresent in a plant genome as a heterozygous trait with a recessivetrait. The plant breeder may decide to produce progeny plants thatpossess the dominant trait. The methods of the disclosure provide asolution to pass the dominant trait into progeny plants. For example, adouble strand break may be specifically introduced into the recessivetrait so that only the dominant trait is passed to the progeny plants.In an embodiment, a first allele may encode a recessive trait and asecond allele may encode a dominant trait. In further embodiments, amethod to introduce a double strand break in the recessive trait resultsin the production of progeny plants that contain the dominant trait.

In further aspects, the locus (including alleles, traits, and/orpolymorphic markers) may be located at a specific position on thechromosome. In some embodiments, the distance between a first locus(including alleles, traits, and/or polymorphic markers) and second locus(including alleles, traits, and/or polymorphic markers) on thechromosome may be spaced so that the first locus is physically separatedfrom the second locus. In an embodiment, the distance between a firstlocus (including alleles, traits, and/or polymorphic markers) and secondlocus (including alleles, traits, and/or polymorphic markers) may rangefrom 0.01 cM to 500 cM. In further embodiments, the distance between afirst locus (including alleles, traits, and/or polymorphic markers) andsecond locus (including alleles, traits, and/or polymorphic markers) maybe at least 0.01 cM. In embodiments, the distance between a first locus(including alleles, traits, and/or polymorphic markers) and second locus(including alleles, traits, and/or polymorphic markers) may be at least0.05 cM. In embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 0.1 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.2 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.3 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.4 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.5 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.6 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.7 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.8 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 0.9 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 1.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 2.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 3.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 4.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 5.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 6.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 7.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 8.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 9.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 10.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 11.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 12.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 13.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 14.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 15.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 16.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 17.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 18.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 19.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 20.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 25.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 30.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 35.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 40.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 45.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 50.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 55.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 60.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 65.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 70.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 75.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 80.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 85.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 90.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 95.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 100.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 125.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 150.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 175.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 200.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 225.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 250.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 275.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 300.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 325.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 350.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 375.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 400.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 425.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 450.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 475.0 cM. Inembodiments, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may be at least 499.0 cM.

In an aspect, the distance between a first locus (including alleles,traits, and/or polymorphic markers) and second locus (including alleles,traits, and/or polymorphic markers) may range from 10 bp to 10 Mbp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 10 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 20 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 30 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 40 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 50 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 60 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 70 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 80 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 90 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 100 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 200 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 300 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 400 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 500 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 600 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 700 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 800 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 900 bp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 1 Kbp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 10 Kbp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 100 Kbp. Infurther embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 1,000 Kbp.In further embodiments, the distance between a first locus (includingalleles, traits, and/or polymorphic markers) and second locus (includingalleles, traits, and/or polymorphic markers) may be at least 1 Mbp.

In traditional genetics, the recombination frequency between twodistinct genetic loci is used as a measure of genetic distance betweenthese loci on a particular chromosome. The maximum frequency ofrecombination between any two loci is 50%, the same value that would beobserved if the genes were on non-homologous chromosomes and assortedindependently. A recombination frequency of 50% occurs when the genesare so far apart on the chromosome that at least one crossing-overalmost always occurs between them. In an aspect, the recombinationfrequency between a first locus (including alleles, traits, and/orpolymorphic markers) and second locus (including alleles, traits, and/orpolymorphic markers) may range from 1% to 50%. In further embodiments,the recombination frequency may be at least 1%. In further embodiments,the recombination frequency may be at least 2%. In further embodiments,the recombination frequency may be at least 3%. In further embodiments,the recombination frequency may be at least 4%. In further embodiments,the recombination frequency may be at least 5%. In further embodiments,the recombination frequency may be at least 6%. In further embodiments,the recombination frequency may be at least 7%. In further embodiments,the recombination frequency may be at least 8%. In further embodiments,the recombination frequency may be at least 9%. In further embodiments,the recombination frequency may be at least 10%. In further embodiments,the recombination frequency may be at least 15%. In further embodiments,the recombination frequency may be at least 20%. In further embodiments,the recombination frequency may be at least 22.5%. In furtherembodiments, the recombination frequency may be at least 25%. In furtherembodiments, the recombination frequency may be at least 27.5%. Infurther embodiments, the recombination frequency may be at least 30%. Infurther embodiments, the recombination frequency may be at least 32.5%.In further embodiments, the recombination frequency may be at least 35%.In further embodiments, the recombination frequency may be at least37.5%. In further embodiments, the recombination frequency may be atleast 40%. In further embodiments, the recombination frequency may be atleast 42.5%. In further embodiments, the recombination frequency may beat least 45%. In further embodiments, the recombination frequency may beat least 47.5%. In further embodiments, the recombination frequency maybe at least 49%.

In further embodiments, the first locus (including alleles, traits,and/or polymorphic markers) may be located at a specific position on afirst chromosome, and the second locus (including alleles, traits,and/or polymorphic markers) on the chromosome may be located at aspecific position on a second chromosome that is non-homologous to thefirst chromosome.

In other embodiments, the first locus (including alleles, traits, and/orpolymorphic markers) may be located at a specific position on a firsthomologous chromosome, and the second locus (including alleles, traits,and/or polymorphic markers) on the chromosome may be located at aspecific position on a second homologous chromosome.

In an aspect, the disclosure relates to a plant genome. As an embodimentof the subject disclosure the genome is contained of homologouschromosomes. During recombination, either in meiosis or mitosis,different loci along the homologous chromosomes will recombine therebyresulting in genetic segregation between the loci. Typically, the locion a homologous chromosome will segregate independently depending uponthe physical spacing between the two loci. The greater the distancebetween the two loci, the greater likelihood that the loci willsegregate in progeny plants. Conversely, if the two loci are locatedclosely to one another, it is less likely that the loci will segregatein progeny plants. Such loci, i.e., that are located closely to oneanother, are described in the art as being genetically linked. It may bedesirable to the plant breeder to increase the frequency of geneticrecombination between two genetically linked loci by introducing adouble strand break in only one of the two homologous chromosomes. In anembodiment, the paternal homologous chromosome is cleaved with a sitespecific nuclease to introduce a double strand break in only thepaternal homologous chromosome, and the maternal homologous chromosomeis not cleaved with a site specific nuclease. In another embodiment, thematernal homologous chromosome is cleaved with a site specific nucleaseto introduce a double strand break in only the maternal homologouschromosome, and the paternal homologous chromosome is not cleaved with asite specific nuclease.

In an aspect, the disclosure relates to a polyploid plant genome. Manyplants possess complex genomes that contain more than two copies ofhomologous chromosomes. For example; maize, tomato, sorghum and rice aretypically diploid; banana and watermelon are typically triploid; durumwheat, cotton and potato are typically tetraploid; bread wheat andkiwifruit are typically hexaploid; and, strawberry and sugarcane aretypically octoploid. It may be desirable to the plant breeder toincrease the frequency of genetic recombination between two geneticallylinked loci by introducing a double strand break in only one of the manyhomologous chromosomes present in a polyploid plant species. In anembodiment, a first chromosome is cleaved with a site specific nucleaseto introduce a double strand break in only one of the many homologouschromosomes present in a polyploid plant species, and the otherhomologous chromosomes are not cleaved with the site specific nuclease.In an embodiment the recombination frequency between the loci locatedclose to the double strand break of the cleaved homologous chromosome isincreased so that the frequency of genetic recombination between twolinked loci is increased as a result of the double strand break that isproduced in only one of the many homologous chromosomes present in apolyploid plant species. In a further embodiment the genetic linkagebetween two linked loci is broken as a result of the double strand breakthat is produced in only one of the many homologous chromosomes presentin a polyploid plant species. In another embodiment the linkage betweentwo linked loci is disrupted as a result of the double strand break thatis produced in only one of the many homologous chromosomes present in apolyploid plant species.

In another aspect, the subject disclosure relates to site specificnuclease for introduction of a double stranded DNA break. In accordancewith one embodiment a site specific nuclease can include a zinc fingernuclease (ZFN) that is used to introduce a double strand break in atargeted genomic locus to facilitate the insertion of a nucleic acid ofinterest. Selection of a target site within the selected genomic locusfor binding by a zinc finger domain can be accomplished, for example,according to the methods disclosed in U.S. Pat. No. 6,453,242, thedisclosure of which is incorporated herein, that also discloses methodsfor designing zinc finger proteins (ZFPs) to bind to a selectedsequence. It will be clear to those skilled in the art that simplevisual inspection of a nucleotide sequence can also be used forselection of a target site. Accordingly, any means for target siteselection can be used in the methods described herein.

For ZFP DNA-binding domains, target sites are generally composed of aplurality of adjacent target subsites. A target subsite refers to thesequence, usually either a nucleotide triplet or a nucleotide quadrupletwhich may overlap by one nucleotide with an adjacent quadruplet that isbound by an individual zinc finger. See, for example, WO 02/077227, thedisclosure of which is incorporated herein. A target site generally hasa length of at least 9 nucleotides and, accordingly, is bound by a zincfinger binding domain comprising at least three zinc fingers. Howeverbinding of, for example, a 4-finger binding domain to a 12-nucleotidetarget site, a 5-finger binding domain to a 15-nucleotide target site ora 6-finger binding domain to an 18-nucleotide target site, is alsopossible. As will be apparent, binding of larger binding domains (e.g.,7-, 8-, 9-finger and more) to longer target sites is also consistentwith the subject disclosure.

In accordance with one embodiment, it is not necessary for a target siteto be a multiple of three nucleotides. In cases in which cross-strandinteractions occur (see, e.g., U.S. Pat. No. 6,453,242 and WO02/077227), one or more of the individual zinc fingers of a multi-fingerbinding domain can bind to overlapping quadruplet subsites. As a result,a three-finger protein can bind a 10-nucleotide sequence, wherein thetenth nucleotide is part of a quadruplet bound by a terminal finger, afour-finger protein can bind a 13-nucleotide sequence, wherein thethirteenth nucleotide is part of a quadruplet bound by a terminalfinger.

The length and nature of amino acid linker sequences between individualzinc fingers in a multi-finger binding domain also affects binding to atarget sequence. For example, the presence of a so-called “non-canonicallinker,” “long linker” or “structured linker” between adjacent zincfingers in a multi-finger binding domain can allow those fingers to bindsubsites which are not immediately adjacent. Non-limiting examples ofsuch linkers are described, for example, in U.S. Pat. No. 6,479,626 andWO 01/53480. Accordingly, one or more subsites, in a target site for azinc finger binding domain, can be separated from each other by 1, 2, 3,4, 5 or more nucleotides. One nonlimiting example would be a four-fingerbinding domain that binds to a 13-nucleotide target site comprising, insequence, two contiguous 3-nucleotide subsites, an interveningnucleotide, and two contiguous triplet subsites.

While DNA-binding polypeptides identified from proteins that exist innature typically bind to a discrete nucleotide sequence or motif (e.g.,a consensus recognition sequence), methods exist and are known in theart for modifying many such DNA-binding polypeptides to recognize adifferent nucleotide sequence or motif. DNA-binding polypeptidesinclude, for example and without limitation: zinc finger DNA-bindingdomains; leucine zippers; UPA DNA-binding domains; GAL4; TAL; LexA; aTet repressor; LacR; and a steroid hormone receptor.

In some examples, a DNA-binding polypeptide is a zinc finger. Individualzinc finger motifs can be designed to target and bind specifically toany of a large range of DNA sites. Canonical Cys₂His₂ (as well asnon-canonical Cys₃His) zinc finger polypeptides bind DNA by inserting anα-helix into the major groove of the target DNA double helix.Recognition of DNA by a zinc finger is modular; each finger contactsprimarily three consecutive base pairs in the target, and a few keyresidues in the polypeptide mediate recognition. By including multiplezinc finger DNA-binding domains in a targeting endonuclease, theDNA-binding specificity of the targeting endonuclease may be furtherincreased (and hence the specificity of any gene regulatory effectsconferred thereby may also be increased). See, e.g., Urnov et al. (2005)Nature 435:646-51. Thus, one or more zinc finger DNA-bindingpolypeptides may be engineered and utilized such that a targetingendonuclease introduced into a host cell interacts with a DNA sequencethat is unique within the genome of the host cell.Preferably, the zincfinger protein is non-naturally occurring in that it is engineered tobind to a target site of choice. See, for example, Beerli et al. (2002)Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-Pspl, PI-Sce, I-SceIV, I-Csml,I-PanI, I-SceII, I-Ppol, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin(1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the NewEngland Biolabs catalogue. In addition, the DNA-binding specificity ofhoming endonucleases and meganucleases can be engineered to bindnon-natural target sites. See, for example, Chevalier et al. (2002)Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128.

As another alternative, the DNA-binding domain may be derived from aleucine zipper protein. Leucine zippers are a class of proteins that areinvolved in protein-protein interactions in many eukaryotic regulatoryproteins that are important transcription factors associated with geneexpression. The leucine zipper refers to a common structural motifshared in these transcriptional factors across several kingdomsincluding animals, plants, yeasts, etc. The leucine zipper is formed bytwo polypeptides (homodimer or heterodimer) that bind to specific DNAsequences in a manner where the leucine residues are evenly spacedthrough an a-helix, such that the leucine residues of the twopolypeptides end up on the same face of the helix. The DNA bindingspecificity of leucine zippers can be utilized in the DNA-bindingdomains disclosed herein.

In some embodiments, the DNA-binding domain is an engineered domain froma TAL effector derived from the plant pathogen Xanthomonas (see, Milleret al. (2011) Nature Biotechnology 29(2):143-8; Boch et al, (2009)Science 29 Oct 2009 (10.1126/science.117881) and Moscou and Bogdanove,(2009) Science 29 Oct 2009 (10.1126/science.1178817; and U.S. PatentPublication Nos. 20110239315, 20110145940 and 20110301073).

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)Cas (CRISPR Associated) nuclease system is a recently engineerednuclease system based on a bacterial system that can be used for genomeengineering. It is based on part of the adaptive immune response of manybacteria and Archea. When a virus or plasmid invades a bacterium,segments of the invader's DNA are converted into CRISPR RNAs (crRNA) bythe ‘immune’ response. This crRNA then associates, through a region ofpartial complementarity, with another type of RNA called tracrRNA toguide the Cas9 nuclease to a region homologous to the crRNA in thetarget DNA called a “protospacer”. Cas9 cleaves the DNA to generateblunt ends at the DSB at sites specified by a 20-nucleotide guidesequence contained within the crRNA transcript. Cas9 requires both thecrRNA and the tracrRNA for site specific DNA recognition and cleavage.This system has now been engineered such that the crRNA and tracrRNA canbe combined into one molecule (the “single guide RNA”), and the crRNAequivalent portion of the single guide RNA can be engineered to guidethe Cas9 nuclease to target any desired sequence (see Jinek et al (2012)Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, and DavidSegal, (2013) eLife 2:e00563). Thus, the CRISPR Cas system can beengineered to create a double-stranded break (DSB) at a desired targetin a genome, and repair of the DSB can be influenced by the use ofrepair inhibitors to cause an increase in error prone repair. OtherCRISPR Cas systems are known in the art and include CRISPR CasX, CRIXPRCasY, CRISP Cpf1, and other similarly functioning enzymes.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein. The Cas protein is deployed in mammalian cells (andputatively within plant cells) by co-expressing the Cas nuclease withguide RNA. Two forms of guide RNAs can be ued to facilitate Cas-mediatedgenome cleavage as disclosed in Le Cong, F., et al., (2013) Science339(6121):819-823.

In other embodiments, the DNA-binding domain may be associated with acleavage (nuclease) domain. For example, homing endonucleases may bemodified in their DNA-binding specificity while retaining nucleasefunction. In addition, zinc finger proteins may also be fused to acleavage domain to form a zinc finger nuclease (ZFN). The cleavagedomain portion of the fusion proteins disclosed herein can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press,1993). Nonlimiting examples of homing endonucleases and meganucleases includeI-SceI, I-CeuI, PI-Pspl, PI-Sce, I-SceIV, I-Csml, I-PanI, I-SceII,I-Ppol, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. Seealso U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997) NucleicAcids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler etal. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast etal. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue. One or more of these enzymes (or functional fragmentsthereof) can be used as a source of cleavage domains and cleavagehalf-domains.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 2007/014275, incorporated by reference herein in itsentirety.

To enhance cleavage specificity, cleavage domains may also be modified.In certain embodiments, variants of the cleavage half-domain areemployed these variants minimize or prevent homodimerization of thecleavage half-domains. Non-limiting examples of such modified cleavagehalf-domains are described in detail in WO 2007/014275, incorporated byreference in its entirety herein. In certain embodiments, the cleavagedomain comprises an engineered cleavage half-domain (also referred to asdimerization domain mutants) that minimize or prevent homodimerization.Such embodiments are known to those of skill the art and described forexample in U.S. Patent Publication Nos. 20050064474; 20060188987;20070305346 and 20080131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of FokI are all targets forinfluencing dimerization of the FokI cleavage half-domains.

Additional engineered cleavage half-domains of FokI that form obligateheterodimers can also be used in the ZFNs described herein. Exemplaryengineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499. In one embodiment, a mutation at 490 replaces Glu(E) with Lys (K); the mutation at 538 replaces Iso (I) with Lys (K); themutation at 486 replaced Gln (Q) with Glu (E); and the mutation atposition 499 replaces Iso (I) with Lys (K). Specifically, the engineeredcleavage half-domains described herein were prepared by mutatingpositions 490 (E→K) and 538 (I→K) in one cleavage half-domain to producean engineered cleavage half-domain designated “E490K:I538K” and bymutating positions 486 (Q→E) and 499 (I→L) in another cleavagehalf-domain to produce an engineered cleavage half-domain designated“Q486E:I499L”. The engineered cleavage half-domains described herein areobligate heterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., U.S. Patent Publication No. 2008/0131962, thedisclosure of which is incorporated by reference in its entirety for allpurposes. In certain embodiments, the engineered cleavage half-domaincomprises mutations at positions 486, 499 and 496 (numbered relative towild-type FokI), for instance mutations that replace the wild type Gln(Q) residue at position 486 with a Glu (E) residue, the wild type Iso(I) residue at position 499 with a Leu (L) residue and the wild-type Asn(N) residue at position 496 with an Asp (D) or Glu (E) residue (alsoreferred to as a “ELD” and “ELE” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490, 538 and 537 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue, the wild type Iso (I) residue atposition 538 with a Lys (K) residue, and the wild-type His (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KKK” and “KKR” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490 and 537 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue and the wild-type His (H) residue atposition 537 with a Lys (K) residue or a Arg (R) residue (also referredto as “KIK” and “KIR” domains, respectively). (See US Patent PublicationNo. 20110201055). In other embodiments, the engineered cleavage halfdomain comprises the “Sharkey” and/or “Sharkey' ” mutations (see Guo etal, (2010) J. Mol. Biol. 400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Distance between target sites refers to the number of nucleotides ornucleotide pairs intervening between two target sites as measured fromthe edges of the sequences nearest each other. In certain embodiments inwhich cleavage depends on the binding of two zinc finger domain/cleavagehalf-domain fusion molecules to separate target sites, the two targetsites can be on opposite DNA strands. In other embodiments, both targetsites are on the same DNA strand. For targeted integration into theoptimal genomic locus, one or more ZFPs are engineered to bind a targetsite at or near the predetermined cleavage site, and a fusion proteincomprising the engineered DNA-binding domain and a cleavage domain isexpressed in the cell. Upon binding of the zinc finger portion of thefusion protein to the target site, the DNA is cleaved, preferably via adouble-stranded break, near the target site by the cleavage domain.

In certain embodiments, two fusion proteins, each comprising aDNA-binding domain and a cleavage half-domain, are expressed in a cell,and bind to target sites which are juxtaposed in such a way that afunctional cleavage domain is reconstituted and DNA is cleaved in thevicinity of the target sites. In one embodiment, cleavage occurs betweenthe target sites of the two DNA-binding domains. One or both of theDNA-binding domains can be engineered. See, also, U.S. Patent No.7,888,121; U.S. Patent Publication 20050064474 and International PatentPublications WO05/084190, WO05/014791 and WO 03/080809.

The site specific nucleases as described herein can be introduced aspolypeptides and/or polynucleotides. For example, two polynucleotides,each comprising sequences encoding one of the aforementionedpolypeptides, can be introduced into a cell, and when the polypeptidesare expressed and each binds to its target sequence, cleavage occurs ator near the target sequence. Alternatively, a single polynucleotidecomprising sequences encoding both fusion polypeptides is introducedinto a cell. Polynucleotides can be DNA, RNA or any modified forms oranalogues or DNA and/or RNA. In an aspect, the subject disclosurerelates to the direct delivery of the site specific nuclease isdelivered to a plant cell. In another aspect, the subject disclosurerelates to the intra-genomic delivery of the site specific nuclease isdelivered to a plant cell.

Through the application of techniques such as these, the cells ofvirtually any species may be stably transformed. In some embodiments,transforming DNA is integrated into the genome of the host cell. In thecase of multicellular species, transgenic cells may be regenerated intoa transgenic organism. Any of these techniques may be used to produce atransgenic plant, for example, comprising one or more donorpolynucleotide acid sequences in the genome of the transgenic plant.

The intra-genomic delivery or the direct delivery of nucleic acids maybe introduced into a plant cell in embodiments of the disclosure by anymethod known to those of skill in the art, including, for example andwithout limitation: by transformation of protoplasts (See, e.g., U.S.Pat. No. 5,508,184); by desiccation/inhibition-mediated DNA uptake (See,e.g., Potrykus et al. (1985) Mol. Gen. Genet. 199:183-8); byelectroporation (See, e.g., U.S. Pat. 5,384,253); by agitation withsilicon carbide fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and5,464,765); by Agrobacterium-mediated transformation (See, e.g., U.S.Pat. Nos. 5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and6,384,301);, by acceleration of DNA-coated particles (See, e.g., U.S.Pat. Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and6,403,865) and by Nanoparticles, nanocarriers and cell penetratingpeptides (WO201126644A2; WO2009046384A1; WO2008148223A1) in the methodsto deliver DNA, RNA, Peptides and/or proteins or combinations of nucleicacids and peptides into plant cells.

The most widely-utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria that genetically transform plant cells. The T_(i) andR_(i) plasmids of A. tumefaciens and A. rhizogenes, respectively, carrygenes responsible for genetic transformation of the plant. The T_(i)(tumor-inducing)-plasmids contain a large segment, known as T-DNA, whichis transferred to transformed plants. Another segment of the T_(i)plasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by left-hand and right-hand borders that are eachcomposed of terminal repeated nucleotide sequences. In some modifiedbinary vectors, the tumor-inducing genes have been deleted, and thefunctions of the vir region are utilized to transfer foreign DNAbordered by the T-DNA border sequences. The T-region may also contain,for example, a selectable marker for efficient recovery of transgenicplants and cells, and a multiple cloning site for inserting sequencesfor transfer such as a nucleic acid encoding a fusion protein of thedisclosure.

Thus, in some embodiments, a plant transformation vector is derived froma Ti plasmid of A. tumefaciens (See, e.g., U.S. Pat. Nos. 4,536,475,4,693,977, 4,886,937, and 5,501,967; and European Patent EP 0 122 791)or a R_(i) plasmid of A. rhizogenes. Additional plant transformationvectors include, for example and without limitation, those described byHerrera-Estrella et al. (1983) Nature 303:209-13; Bevan et al. (1983),supra; Klee et al. (1985) Bio/Technol. 3:637-42; and in European PatentEP 0 120 516, and those derived from any of the foregoing. Otherbacteria, such as Sinorhizobium, Rhizobium, and Mesorhizobium thatnaturally interact with plants can be modified to mediate gene transferto a number of diverse plants. These plant-associated symbiotic bacteriacan be made competent for gene transfer by acquisition of both adisarmed Ti plasmid and a suitable binary vector.

In addition, once a site specific nuclease has been stably integratedinto the genome of a first plant, the nuclease can be bred into aprogeny plant and deployed to bind and cleave a specific target sight bycrossing the first and second parent plant. In such an example, thegenome of the first parent plant does not contain a target sequence thatis bound and cleaved by a site specific nuclease. However, the secondparent plant does contain a target sequence that is bound and cleaved bythe site specific nuclease located in the first parent plant.Accordingly, the breeding of the first and second parent plant allowsfor the site specific nuclease of the first parent plant to befunctional in progeny plants that inherit both the site specificnuclease from the first parent plant and the target sequence of thesecond parent plant. In such an embodiment, the site specific nucleaseis delivered to a plant cell by intra-genomic recombination.

In an aspect of the subject disclosure, the methods provided areapplicable foe producing a progeny plant comprising a modified genome.The development of progeny plants is typically achieved throughconventional plant breeding techniques. Such breeding techniques arewell known to one skilled in the art. For a discussion of plant breedingtechniques, see Poehlman (1995) Breeding Field Crops. AVI PublicationCo., Westport Conn, 4^(th) Edit, herein incorporated by reference in itsentirety. Backcrossing methods may be used to introduce a gene into theplants. This technique has been used for decades to introduce traitsinto a plant. An example of a description of this and other plantbreeding methodologies that are well known can be found in referencessuch as Poelman, supra, and Plant Breeding Methodology, edit. NealJensen, John Wiley & Sons, Inc. (1988). In a typical backcross protocol,the original variety of interest (recurrent parent) is crossed to asecond variety (nonrecurrent parent) that carries the single gene ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

Certain embodiments relate to processes of making crosses using a plantof an embodiment of this disclosure as at least one parent. For example,particular embodiments relate to an F₁ hybrid plant having as one orboth parents any of the plants exemplified herein. Other embodimentsrelate to seed produced by such F₁ hybrids. Still other embodimentsrelate to a method for producing an F₁ hybrid seed by crossing anexemplified plant with a different (e.g. in-bred parent) plant andharvesting the resultant hybrid seed. Other embodiments relate to anexemplified plant that is either a female parent or a male parent.Characteristics of the resulting plants may be improved by carefulconsideration of the parent plants.

As an embodiment of the subject disclosure, a progeny plant can be bredby first sexually crossing a first parental plant and a second parentalplant, thereby producing a plurality of first progeny plants; thenselecting a first progeny plant that contains the loci of interestunlinked to the second loci; selfing the first progeny plant, therebyproducing a plurality of second progeny plants; and then selecting fromthe second progeny plants a plant that contains the loci of interestunlinked to the second loci. These steps can further include theback-crossing of the first progeny plant or the second progeny plant tothe second parental plant or a third parental plant. A crop comprisingseeds of particular embodiments, or progeny thereof, can then beplanted.

It is also to be understood that two plants can also be crossed toproduce offspring that contain independently segregating genes as aresult of the disclosed method. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with another plantare also contemplated, as is vegetative propagation. Other breedingmethods commonly used for different traits and crops are known in theart. Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

Methodologies for regenerating plants are known to those of ordinaryskill in the art and can be found, for example, in: Plant Cell andTissue Culture, 1994, Vasil and Thorpe Eds. Kluwer Academic Publishersand in: Plant Cell Culture Protocols (Methods in Molecular Biology 111,1999 Hall Eds Humana Press). The plant described herein can be culturedin a fermentation medium or grown in a suitable medium such as soil. Insome embodiments, a suitable growth medium for higher plants can includeany growth medium for plants, including, but not limited to, soil, sand,any other particulate media that support root growth (e.g., vermiculite,perlite, etc.) or hydroponic culture, as well as suitable light, waterand nutritional supplements which optimize the growth of the higherplant.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.

In embodiments, the present disclosure relates to regenerable cells foruse in tissue culture of. The tissue culture will preferably be capableof regenerating plants having the physiological and morphologicalcharacteristics of the foregoing plants and of regenerating plantshaving substantially the same genotype as the foregoing plants.Preferably, the regenerable cells in such tissue cultures will beembryos, protoplasts, meristematic cells, callus, pollen, leaves,anthers, roots, root tips, flowers, seeds, pods or stems. Still further,embodiments of the present disclosure relate to plants regenerated fromthe tissue cultures of embodiments of the disclosure.

In an aspect of the subject disclosure, the methods provided areapplicable on a wide variety of plants and plant cell systems. Inembodiments, target plants and plant cells for engineering include, butare not limited to, those monocotyledonous and dicotyledonous plants,such as crops including grain crops (e.g., wheat, maize, rice, millet,barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange),forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot,potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce,spinach); flowering plants (e.g., petunia, rose, chrysanthemum),conifers and pine trees (e.g., pine fir, spruce); plants used inphytoremediation (e.g., heavy metal accumulating plants); oil crops(e.g., sunflower, rape seed) and plants used for experimental purposes(e.g., Arabidopsis). Thus, the disclosed methods and compositions haveuse over a broad range of plants, including, but not limited to, speciesfrom the genera Asparagus, Avena, Brassica, Citrus, Citrullus, Capsicum,Cucurbita, Daucus, Erigeron, Glycine, Gossypium, Hordeum, Lactuca,Lolium, Lycopersicon, Malus, Manihot, Nicotiana, Orychophragmus, Oryza,Persea, Phaseolus, Pisum, Pyrus, Prunus, Raphanus, Secale, Solanum,Sorghum, Triticum, Vitis, Vigna, and Zea mays.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1 Design of Gene Expression Construct

The Engineered Transgene Integration Platform (ETIP) was previouslydescribed in U.S. Patent Application No. 20140090113A1, hereinincorporated by reference in its entirety. The ETIP contains anengineered landing pad sequence, several engineered zinc finger bindingsites (eZFN), and gene expression cassettes as shown in the binaryplasmid vector pDAB105855 (FIG. 1; SEQ ID NO:1). As described in U.S.Patent Application No. 20140090113A1, the binary plasmid, pDAB105855,was transformed and integrated into the genome of Zea mays c.v. B104(see, Example 8 of U.S. Patent Application No. 20140090113A1). Positivetransgenic plants containing full-length, simple insertion events of thepDAB105855 T-strand insert were confirmed via molecular analysis (see,Example 6 of U.S. Patent Application No. 20140090113A1). The transgeneexpression cassettes within the Agrobacterium border regions ofpDAB105855 were located within the chromosome of the Zea mays c.v. B104genome as a full length T-strand insert. The resulting transgenic plantcontaining transgenic event, pDAB105855-#2, was self-pollinated toproduce homozygous progeny plants that contained full length copies ofthe T-strand insert from pDAB105855. These progeny plants were confirmedas being homozygous by molecular confirmation assays.

Example 2 Design of Gene Expression Construct

A second gene expression construct containing the dgt-28 transgene andseveral eZFN binding sites was designed and built. This gene expressionconstruct was labeled as pDAB113068 (FIG. 2; SEQ ID NO:2) and containedthe following gene elements: the eZFN1 Binding Site; the Oryza sativaUbiquitin 3 intron (OsUbi3 intron; Sivamani, E., Qu, R., (2006) PlantMolecular Biology 60; 225-239) driving the expression of the dgt-28transgene (DGT-28; International Patent Application No. 2013116700)fused with the TraP4 chloroplast transit peptide (International PatentApplication No. 2013158766) and terminated by the Zea mays lipase 3′UTR(ZmLip 3′UTR; U.S. Pat. No. 7,179,902); a linker region containingmultiple site specific nuclease binding sites (SBS8196 BindingSite::eZFN4 Binding Site::SBS19354 Binding Site::SBS15590 BindingSite::eZFN8 Binding Site::SBS18473 Binding Site::eZFN1 Binding Site);and, the Zea mays Ubiquitin1 promoter (ZmUbi1 promoter; Christensen etal. (1992) Plant Molecular Biology 18; 675-689) driving the expressionof the pat transgene (PAT; Wohlleben et al. (1988) Gene 70(1); 25-37)and terminated by the Zea mays lipase 3′UTR. This gene expressionconstruct was transformed into the pDAB105855-#2 event Zea mays plantsthat were previously confirmed to contain homozygous copies ofpDAB105855.

Example 3 Agrobacterium Strain Production and Zea Mays Transformation

Inoculation of Agrobacterium tumefaciens

The pDAB113068 binary plasmid was transformed into the pDAB105855-#2event Zea mays plants via an Agrobacterium mediated transformationprotocol. The binary expression vectors were transformed intoAgrobacterium tumefaciens strain EHA105. Bacterial colonies wereselected, and binary plasmid DNA was isolated and confirmed viarestriction enzyme digestion. The Agrobacterium cultures were streakedfrom glycerol stocks and incubated for growth. On the day of anexperiment, the resulting cultures of Agrobacterium were used for thetransformation of Zea mays plants containing the pDAB105855-#2 event.

Zea Mays Transformation

Experimental constructs were transformed into Zea mays plants containingthe pDAB105855-#2 event via Agrobacterium-mediated transformation ofimmature embryos isolated from the inbred line, Zea mays c.v. B104plants containing the pDAB105855-#2 event. The method used is similar tothose published by Ishida et al. (1996) Nature Biotechnol 14:745-750 andFrame et al. (2006) Plant Cell Rep 25: 1024-1034, but with severalmodifications and improvements to make the method amenable tohigh-throughput transformation. An example of a method used to produce anumber of transgenic events in Zea mays is given in U.S. PatentApplication No. 20130157369A1, beginning with the embryo infection andco-cultivation steps.

Example 4 Molecular Confirmation of Copy Number at T0

Putative transgenic Zea mays plants were sampled at the V2-3 leafdevelopment stage for transgene presence using a pat transgenequantitative PCR assay. Total DNA was extracted from leaf punches usingMagAttract® DNA extraction kit (Qiagen) as per the manufacturer'sinstruction.

To detect the genes of interest, gene-specific DNA fragments wereamplified with TaqMan® primer/probe sets containing a FAM-labeledfluorescent probe for the pat transgene and a HEX-labeled fluorescentprobe for an endogenous reference gene control. Next, the PCR reactionswere carried out in a final volume of 10 μl reaction containing 5 μl ofRoche LightCycler® 480 Probes Master Mix (Roche Applied Sciences,Indianapolis, Ind.); 0.4 μl each of the primers from 10 μM stocks to afinal concentration of 400 nM; 0.4 μl each of the probes from 5 μMstocks to a final concentration of 200 nM, 0.1 μl of 10%polyvinylpyrrolidone (PVP) to final concentration of 0.1%; 2 μl of 10ng/μl genomic DNA and 0.5 μl water. The DNA was amplified in a RocheLightCycler® 480 System under the following conditions: 1 cycle of 95°C. for 10 min; 40 cycles of the following 3-steps: 95° C. for 10seconds; 58° C. for 35 seconds and 72° C. for 1 second, and a finalcycle of 4° C. for 10 seconds. The pat transgene copy number wasdetermined by comparison of Target (gene of interest)/Reference valuesfor unknown samples (output by the LightCycler® 480) to Target/Referencevalues of the pat transgene copy number controls. The resulting T0plants were selfed to obtain T1 seed, which was screened to identifytransgenic plants that were homozygous. Zygosity screening was completedon the T1 progeny plants to identify the plants that were homozygous forboth the target and donor gene events. These progeny plants wereutilized for the additional experiments.

Example 5 Mapping of Chromosomal Location of the Events

The transgenic plant containing target pDAB105822-#2 event, and donorpDAB113068-#250 event were analyzed using Next Generation Sequencing(NGS) to determine the genomic insertion site locations of bothtransgenes. The sequence reads were aligned to the Zea mays B73reference genome from maizeGDB (Andorf et al. (2015) MaizeGDB 2015: Newtools, data, and interface for the maize model organism database.Nucleic Acids Research doi: 10.1093/nar/gkv1007). Target pDAB105822#2and donor pDAB113068-#250 event were found to be inserted on the samechromosome. The target pDAB105855-#2 event was mapped to Chromosome 5:188,528,595..188,528,607, while the donor pDAB113068-#250 event wasmapped to Chromosome 5: 147,625,397..147,625,455.

Example 6 Design of ZFNs and Gene Expression Construct

Zinc finger proteins directed against the eZFN1 DNA sequence whichcomprise a site specific cleavage site within the T-DNA of pDAB105855(see, FIG. 1) were designed as previously described. See, e.g., Urnov etal. (2005) Nature 435:646-651. Exemplary recognition helices werepreviously disclosed as “recognition helix region designs” in U.S.Patent Application No. 20140090113A1 (herein incorporated by referencein its entirety). The target sequence site that the zinc fingerrecognized and bound is provided in Table 1 as SEQ ID NO:3.

TABLE 1 Site specific nuclease target sequence SEQ ID Site Name NO:Target Sequence eZFN1 Binding Site 3 caatcctgtccctagtg gataaactgcaaaaggc

The site specific nuclease zinc finger designs were incorporated intovectors encoding a protein having at least one finger with a CCHCstructure. See, U.S. Patent Application No. 20080182332. In particular,the last finger in each protein had a CCHC backbone for the recognitionhelix. The non-canonical zinc finger-encoding sequences were fused tothe nuclease domain of the type IIS restriction enzyme FokI (amino acids384-579 of the sequence of Wah et al. (1998) Proc. Natl. Acad. Sci. USA95:10564-10569) via a four amino acid ZC linker and an opaque-2 nuclearlocalization signal derived from Zea mays to form eZFN1 specificzinc-finger nucleases (ZFNs). Expression of the fusion proteins in abicistronic expression construct utilizing a 2A ribosomal stutteringsignal as described in Shukla et al. (2009) Nature 459:437-441 wasdriven by a relatively strong, constitutive Zea mays Ubiquitin 1promoter.

The optimal zinc fingers were verified for cleavage activity using abudding yeast based system previously shown to identify activenucleases. See, e.g., U.S. Patent Publication No. 20090111119; Doyon etal. (2008) Nat Biotechnol. 26:702-708; Geurts et al. (2009) Science325:433. Zinc fingers for the various functional domains were selectedfor in-vivo use. Of the numerous ZFNs that were designed, produced andtested to bind to the putative eZFN1 polynucleotide target sites, a pairof ZFNs were identified as having in vivo activity at high levels, andselected for further experimentation. These ZFNs were characterized asbeing capable of efficiently binding and cleaving the site specificeZFN1 genomic polynucleotide target sites in planta. After testing theZFN pairs in the budding yeast assay, ZFN pairs which optimally boundthe eZFN1 binding site were advanced for testing in Zea mays.

Zinc finger nuclease constructs for expression in Zea mays;

Plasmid vectors containing ZFN expression constructs of the exemplaryzinc finger nucleases, which were identified using the yeast assay anddescribed above, were designed and completed using skills and techniquescommonly known in the art. Each zinc finger-encoding sequence was fusedto a sequence encoding an opaque-2 nuclear localization signal(Maddaloni et al. (1989) Nuc. Acids Res. 17(18):7532), that waspositioned upstream of the zinc finger nuclease.

Next, the opaque-2 nuclear localization signal::zinc finger nucleasefusion sequence was paired with the complementary opaque-2 nuclearlocalization signal::zinc finger nuclease fusion sequence. As such, eachconstruct consisted of a single open reading frame comprised of twoopaque-2 nuclear localization signal::zinc finger nuclease fusionsequences separated by the 2A sequence from Thosea asigna virus (Mattionet al. (1996) J. Virol. 70:8124-8127). Expression of the ZFN codingsequence was driven by the highly expressing constitutive Zea maysUbiquitin 1 Promoter (Christensen et al. (1992) Plant Mol. Biol.18(4):675-89) and flanked by the Zea mays Per 5 3′ polyA untranslatedregion (U.S. Pat. No. 6,699,984). The resulting four plasmid constructswere confirmed via restriction enzyme digestion and via DNA sequencing.FIG. 3 provides a graphical representation of the completed plasmidconstruct of pDAB105825. The ZFN expressed in plasmid construct,pDAB105825, contains “Fok-Mono” which is a wildtype FokI endonuclease.

Example 7 Zea Mays Transformation

The resulting zinc finger nuclease construct, pDAB105825, wastransformed into Zea mays c.v. B104 plants using the methods previouslydescribed in Example 3 above. The resulting transgenic plant containingwas self-pollinated to produce homozygous progeny plants that containedfull length copies of the T-strand insert from pDAB105825. These progenyplants were confirmed as being homozygous by molecular confirmationassays as described in Example 4 above.

Example 8 Crossing of the Homozygous T1 Plants for Producing an F1Population

The T1 pDAB105825 events were screened for zygosity and expression ofthe aad-1 transgene. Based on these results, homozygous T1 plants fromone pDAB105825 event were selected for crossing withpDAB105855/pDAB113068 plant events to produce F1 progeny plants.Reciprocal crosses were made so that parents were both male and female.The plants were crossed by hand; pollen from the anthers of a maturemale parent was introduced to the stigma of the mature female parent.Plants ready for crossing were removed from the other plants to reducethe likelihood that unintended pollen would fertilize the female maizeplants. Female plants were emasculated (anthers removed prior todehiscence) by detasseling. The anthers from the male parent weretotally removed from the male plant, and the pollen was isolated fromthe anthers and used to fertilize the emasculated female. The isolatedpollen was rubbed onto the receptive silks of female plants, coating thesilks to reduce the chance of any unintended pollinations. The seed fromthe fertilized plants was collected and sewn into soil. The resulting F1populations were grown in the greenhouse under standard maize growingconditions.

Example 9 Crossing of the F1 Plants to Sibling Null Plants to Produce aBC1 Population

The resulting F1 progeny plants that contained all three events (i.e.,pDAB105855/pDAB113068/pDAB105825) were grown to maturity and backcrossedto sibling null parent plants to produce a BC1 population. The crossingstrategy is set forth below and diagrammed in FIG. 4. The resulting BC1populations were molecularly characterized to calculate therecombination frequencies which occurred between the genetically linkedtransgenes between the pDAB105855 gene and the pDAB113068 gene. As aresult of cleaving one of two homologous chromosomes with zinc fingernucleases, it was hypothesized that the recombination frequency betweenthe pDAB105855 gene and the pDAB113068 gene would increase as the resultof the introduction of a double strand break at the eZFN1 binding siteby a zinc finger nuclease FIG. 5.

Example 10 Crossing of the BC1 Plants to Sibling Null Plants to Producea BC2 Population

The resulting BC1 progeny plants that contained all three events (i.e.,pDAB105855/pDAB113068/pDAB105825) were grown to maturity and backcrossedto sibling null parent plants to produce a BC2 population. The crossingstrategy is set forth below and diagrammed in FIG. 4. The resulting BC2populations were molecularly characterized to calculate therecombination frequencies which occurred between the genetically linkedtransgenes; the pDAB105855 gene and the pDAB113068 gene. As a result ofcleaving one of two homologous chromosomes with zinc finger nucleases,it was hypothesized that the recombination frequency between thepDAB105855 gene and the pDAB113068 gene would increase as the result ofthe introduction of a double strand break at the eZFN1 binding site by azinc finger nuclease FIG. 5.

Example 11 Calculation of the Percentage of Recombination Frequency

The recombination frequency between the genetically linked transgenes,for instance the pDAB105855 gene and the pDAB113068 gene, was calculatedand is provided in Table 2. Gene 1 and 2 are genetically linked andtherefore frequently co-segregate. The percentage of recombinationfrequency was determined by dividing the number of recombinant offspring(containing gene 1 or gene 2 alone) by the total number of offspringobserved.

The control progeny plants, which did not include the introduction of azinc finger nuclease within the plant crossing experiments resulted in alow percentage of recombination between the pDAB105855 gene and thepDAB113068 gene was calculated at 3.5%. These control plants did notcontain a double strand break between the genetically linked transgenesthe pDAB105855 gene and the pDAB113068 gene and any segregationoccurring between the pDAB105855 gene and the pDAB113068 gene resultedfrom natural cell processes occurring during the various cell cycles andplant development phases.

Comparatively, the recombination frequency increased by up to 62.3% inthe experimental progeny plants, and the recombination frequency rangedfrom 4.4% to 62.3%. The experimental progeny plants, which included theintroduction of a zinc finger nuclease within the plant crossingexperiments resulted in a higher percentage of recombination between thepDAB 105855 gene and the pDAB 113068 gene was calculated was muchgreater than 3.5% recombination frequency of the control plants. Theseexperimental plants did contain a double strand break between thegenetically linked transgenes of the pDAB105855 gene and the pDAB113068gene, and the increase in segregation occurring between the pDAB105855gene and the pDAB113068 gene resulted from the introduction of thedouble strand break. The frequency of recombinant offspring wasincreased dramatically (up to 62%) with the introduction of a ZFN andthe induction of targeted double strand break at a binding site locatedbetween two genetically linked genes; the pDAB 105855 gene and thepDAB113068 gene.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

TABLE 2 Recombination Frequency in Progeny Plants after Crossing BetweenParental Plants to Introduce a Double Strand Break Between GeneticallyLinked Genes. pDAB105855/ Cross/ Total Null/ pDAB113068/ pDAB113068/pDAB10585/ Recombinant Percentage of Generation Offspring pDAB105825pDAB105825 pDAB105825 pDAB105825 Offspring Recombination Control BC1 5735 20 1 1 2 3.5% 1ZFN BC1 68 62 3 2 1 3 4.4% 1ZFN BC2 61 29 25 3 4 711.5% 2ZFN BC1 70 31 34 4 1 5 7.1% 2ZFN BC2 68 37 28 3 0 3 4.4% 3ZFN BC153 28 9 0 16 16 30.2% 3ZFN BC2 63 29 28 4 1 5 7.9% 4ZFN BC1 69 26 0 0 4343 62.3% 4ZFN BC2 57 31 0 0 26 26 45.6% 5ZFN BC1 69 23 41 2 3 5 7.2%5ZFN BC2 72 21 31 12 8 20 27.8%

What is claimed is:
 1. A method for increasing the frequency of geneticrecombination between a first locus genetically linked to a second locuswithin a genome of a plant, the method comprising the steps of: a)introducing a site specific nuclease into the genome of the plant; b)producing a double stranded break with the site specific nuclease in oneof two homologous chromosomes; c) undergoing recombination within theplant genome; and, d) modifying the plant genome, wherein the modifiedplant genome comprises increased frequency of genetic recombinationbetween the first locus and the second locus.
 2. The method of claim 1,wherein the first locus and second locus encode at least one trait. 3.The method of claim 1, wherein recombination comprises meioticrecombination or mitotic recombination.
 4. The method of claim 1,wherein the increased frequency of genetic recombination ranges from1.25 to 17.8 fold.
 5. The method of claim 1, wherein the distance fromthe first locus to the second locus ranges from about 0.01 cM to about500 cM.
 6. The method of claim 1, wherein the distance from the firstlocus to the second locus ranges from about 10 bp to about 10 Mbp. 7.The method of claim 1, wherein the first locus is located on a firstchromosome, and the second locus is located on a second chromosome. 8.The method of claim 1, wherein the first locus and the second locus arepresent in a genomic location with low levels of recombinationfrequency.
 9. The method of claim 2, wherein the trait comprises adesirable trait or an undesirable trait.
 10. The method of claim 9,wherein the desirable trait or the undesirable trait is either a nativetrait or a transgenic trait.
 11. The method of claim 10, wherein theundesirable trait is selected from the group consisting of reducedyield, reduced resistance to disease, reduced resistance to pests,reduced tolerance to herbicide tolerance, reduced growth, reduced size,reduced production of biomass, reduced amount of produced seeds, reducedresistance against salinity, reduced resistance against heat stress,reduced resistance against cold stress, reduced resistance againstdrought stress, and any combination thereof.
 12. The method of claim 10,wherein the desirable trait is selected from the group consisting ofincreased yield, increased resistance to disease, increased resistanceto pests, increased tolerance to herbicides, increased growth, increasedsize, increased production of biomass, increased amount of producedseeds, increased resistance against salinity, increased resistanceagainst heat stress, increased resistance against cold stress, increasedresistance against drought stress, and any combination thereof.
 13. Themethod of claim 1, wherein the first locus comprises a polymorphicmarker and the second locus comprise a trait.
 14. The method of claim 1,wherein the first locus comprises a polymorphic marker and the secondlocus comprises a polymorphic marker.
 15. The method of claim 1, whereinthe double stranded break is produced by a site specific nuclease in oneof two homologous chromosomes, and a double stranded break is notproduced in the second homologous chromosome.
 16. The method of claim 1,wherein the site specific nuclease is selected from the group consistingof a zinc finger nuclease, a TALEN nuclease, a CRISPR nuclease, ameganuclease, and a leucine zipper nuclease.
 17. The method of claim 1,wherein the site specific nuclease is delivered to a cell byintra-genomic recombination or via direct delivery.
 18. The method ofclaim 1, wherein the genome of the plant is a polyploid.
 19. The methodof claim 1, the method further comprising the steps of: e) producing aprogeny plant comprising the modified plant genome; f) crossing theprogeny plant with another plant or to itself; and, g) generating a seedfrom the progeny plant.
 20. The method of claim 2, wherein the firstlocus comprising a first trait is located on the first homologouschromosome and the second locus comprising a second trait is located onthe first homologous chromosome.
 21. The method of claim 20, wherein theresulting double strand break occurs between the first locus comprisingthe first trait and the second locus comprising the second trait,thereby resulting in a progeny plant comprising only the first locuscomprising the first trait.
 22. The method of claim 2, wherein the firsttrait is either a recessive or a dominant trait.
 23. The method of claim2, wherein the first trait is either a heterozygous or a homozygoustrait.
 24. The method of claim 1, wherein the plant is selected from adicotyledonous plant or a monocotyledonous plant.
 25. The method ofclaim 24, wherein the plant is selected from the group consisting of atobacco plant, a soybean plant, a cotton plant, a Brassica plant, a cornplant, a sorghum plant, a wheat plant, and a rice plant.